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Studies of some mutants of human hemoglobin including a new oC-variant: Hb mahidol Pootrakul, Sanga 1970

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STUDIES OF SOME MUTANTS OF HUMAN HEMOGLOBIN INCLUDING A NEW a-VARIANT: Hb MAHIDOL by SA-NGA POOTRAKUL M.D., U n i v e r s i t y o f M e d i c a l S c i e n c e s , T h a i l a n d , 1962 Graduate Diploma i n C l i n i c a l S c i e n c e (Med.), U n i v e r s i t y o f M e d i c a l S c i e n c e s , T h a i l a n d , 1964 M.Sc. (Med.), U n i v e r s i t y o f M e d i c a l S c i e n c e s , T h a i l a n d , 1966 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Pathology We accept t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d : THE UNIVERSITY OF BRITISH COLUMBIA JUNE, 19 70 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r equ i r emen t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag ree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t hou t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada i ABSTRACT STUDIES OF SOME MUTANTS OF HUMAN HEMOGLOBIN INCLUDING A NEW a VARIANT: Hb MAHIDOL Hemoglobinopathies, the d i s o r d e r s of hemoglobin s t r u c t u r e and s y n t h e s i s , can be d i v i d e d i n t o two forms o f c l i n i c a l m a n i f e s t a t i o n , namely t h a l a s s e m i a s and abnormal hemoglobins. Thalassemia appears to i n v o l v e an abnormal gene which r e s u l t s i n a reduced r a t e o f g l o b i n s y n t h e s i s and i t presents c l i n i c a l l y as a hypochromic m i c r o c y t i c anemia. I t s cause l i e s i n some abnormality of the r e g u l a t i o n o f g l o b i n synthesis. 0 An abnormal hemoglobin i s u s u a l l y the r e s u l t o f a mutation of one base i n a codon t r i p l e t o f the s t r u c t u r a l gene f o r one or o t h e r hemoglobin c h a i n which leads t o an amino a c i d s u b s t i t u t i o n i n the primary s t r u c t u r e of the g l o b i n . In t h i s study, the b i o c h e m i c a l c h a r a c t e r i z a t i o n of f i v e samples of abnormal hemoglobin which were obtained from p a t i e n t s a t Vancouver General H o s p i t a l , Vancouver, B.C. and S i r i r a j H o s p i t a l , Bangkok i s d e s c r i b e d . In the f i r s t case from V.G.H., the p r o p o s i t u s , a 17 month o l d Chinese g i r l showed a f a s t (anionic) abnormal hemoglobin from b i r t h . In a b i o c h e m i c a l i n v e s t i g a t i o n o f the v a r i a n t from her f a t h e r i t was found t h a t the g l y c i n e r e s i d u e 56 i n the 3-chain was s u b s t i t u t e d by a s p a r t i c a c i d . T h i s mutant i s i d e n t i c a l t o t h a t p r e v i o u s l y d e s i g n a t e d as HbJ Bangkok (a A$| 6 A s p). i i The second case, a 21 y e a r o l d T h a i male i n Bangkok was shown t o be a double heterozygote w i t h both a slow and a f a s t abnormal hemoglobin and an absence o f HbA upon s t a r c h g e l e l e c t r o p h o r e s i s . S t r u c t u r a l c h a r a c t e r i z a t i o n of the s e p a r a t e d g l o b i n c h a i n of both v a r i a n t s i n d i c a t e d t h a t the mutation i n the slow v a r i a n t o c c u r r e d a t r e s i d u e 26 of g-chain where glutamic a c i d was r e p l a c e d by l y s i n e . T h i s mutation i s s i m i l a r to t h a t p r e v i o u s l y d e s c r i b e d as HbE ( a ^ 3 § 6 L y S ) . The f a s t v a r i a n t showed an amino a c i d a l t e r a t i o n a t p o s i t i o n 113 of 3-chain where v a l i n e was s u b s t i t u t e d by glutamic a c i d . T h i s mutation i s i d e n t i c a l w i t h Hb New York ( a 2 3 i l s G ; l u ) . A double heterozygote i n v o l v i n g both HbE and Hb New York has not been p r e v i o u s l y d e s c r i b e d . The other three samples from u n r e l a t e d p a t i e n t s i n Bangkok r e v e a l e d a slow mutant on s t a r c h g e l e l e c t r o p h o r e s i s and b i o c h e m i c a l s t u d i e s showed t h a t r e s i d u e 74 i n the a-chain was changed from an a s p a r t y l to a h i s t i d y l r e s i d u e . T h i s mutation has not been p r e v i o u s l y d e s c r i b e d . I t i s proposed t h a t t h i s new hemoglobin (aJ' | H l s3^) be c a l l e d Hb Mahidol a f t e r Mahidol U n i v e r s i t y i n Bangkok. In one o f the t h r e e p a t i e n t s showing Hb Mahidol i n t e r a c t i o n w i t h a-thalassemia (a-thalassemiaLHb Mahidol) o c c u r s . T h i s r e s u l t s i n the c l i n i c a l f e a t u r e s of c h r o n i c h e m o l y t i c anemia and a t o t a l absence of HbA which i s r e p l a c e d by Hb Mahidol t o g e t h e r with some HbH(g^). i i i TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES i x LIST OF FIGURES x i LIST OF ABBREVIATIONS XV ACKNOWLEDGEMENT x v i DEDICATION x v i i INTRODUCTION 1 I . HEMOGLOBIN: STRUCTURE AND GENETICS 3 A. Normal Hemoglobins 3 1. A d u l t hemoglobins a. Hemoglobin A 3 b. Hemoglobin A 2 4 c. Hemoglobin A 3 4 2. F e t a l hemoglobin 4 3. Embryonic hemoglobins 6 a. Hemoglobin - Gower I 6 b. Hemoglobin -Gower I I 6 B. Development of G l o b i n Chains 7 C. Hemoglobin S t r u c t u r e 9 1. S t a b i l i t y o f the hemoglobin molecule 11 a. Hydrophobic bonds 12 b. Heme b i n d i n g 12 c. Sub-unit i n t e r a c t i o n s 12 i . U n l i k e c h a i n i n t e r a c t i o n s 15 i i . L i k e c h a i n i n t e r a c t i o n s 18 2. D i s s o c i a t i o n o f sub-units 18 i v Page I I . HEMOGLOBINOPATHIES 20 A. Nomenclature of Abnormal Hemoglobins 20 B. Abnormal Hemoglobins 1. H e r e d i t a r y abnormal hemoglobins 22 a. Amino a c i d s u b s t i t u t i o n 22 i . S i n g l e amino a c i d s u b s t i t u t i o n 22 a Alpha c h a i n 22 b) Beta c h a i n 24 o) Gamma c h a i n 28 d) D e l t a c h a i n 28 i i . Two amino a c i d s u b s t i t u t i o n s i n p o l y p e p t i d e c h a i n 28 b. P o s s i b l e abnormality d u r i n g meiosis c. Chain aggregations due t o alpha c h a i n d e f i c i e n c y 29 d. Other abnormal hemoglobin 29 2. A c q u i r e d abnormal hemoglobins 29 C. How the S t r u c t u r e o f Abnormal Hemoglobins R e l a t e s t o T h e i r M o l e c u l a r Pathology 30 1. Amino a c i d a l t e r a t i o n i n the r e g i o n o f the heme group 31 a. Replacement o f h i s t i d i n e 31 b. Replacement o f non-polar c o n t a c t s s u r r o u n d i n g the heme 33 2. Amino a c i d a l t e r a t i o n s a t s u b - u n i t contacts 34 a. Amino a c i d replacement a t the a 1-3 2 c o n t a c t 34 b. Amino a c i d replacement a t the a 1 - ^ 1 c o n t a c t 35 V Page 3. Amino a c i d a l t e r a t i o n a t i n t e r n a l p o s i t i o n s i n the hemoglobin molecule 36 4. Amino a c i d a l t e r a t i o n on the o u t s i d e of hemoglobin molecule 37 D. Thalassemias 1. D e s i g n a t i o n o f thalassemias 38 a. H e m a t o l o g i c a l evidence 39 b. B i o c h e m i c a l evidence 39 c. Genetic evidence 40 2. a-thalassemias 40 a. a - t h a l a s s e m i a i ( c l a s s i c a l a-thalassemia) 40 b. a - t h a l a s s e m i a 2 ( m i l d a-thalassemia) 41 3. p - t h a l a s s e m i a s 41 a. A 2 - t h a l a s s e m i a 41 b. F-thalassemia 42 E. Combinations o f Abnormal Genes 42 I I I . MATERIALS AND METHODS 46 A. Hemolysate P r e p a r a t i o n 46 B. S t a r c h Gel E l e c t r o p h o r e s i s 46 C. Hemoglobin Chain D i s s o c i a t i o n With p-Hydroxymercuribenzoate' (PHMB) 4 8 D. G l o b i n P r e p a r a t i o n 49 E. G l o b i n Chain S e p a r a t i o n 49 F. A m i n o e t h y l a t i o n o f Separated G l o b i n Chains 51 G. T r y p t i c D i g e s t i o n 52 H. Peptide Mapping 53 v i Page I . P e p t i d e S t a i n i n g 53 1. S t a i n i n g f o r methionine 53 2. Pauly s t a i n f o r h i s t i d i n e and t y r o s i n e 54 3. T y r o s i n e s t a i n 54 4. Tryptophan s t a i n 54 5. Sakaguchi s t a i n 55 6. N i n h y d r i n and cadmium n i n h y d r i n s t a i n 55 J . I s o l a t i o n o f Pepti d e s on a P r e p a r a t i v e S c a l e 56 K. Cyanogen Bromide Cleavage o f Methionine C o n t a i n i n g Peptides 57 L. D i l u t e A c e t i c Cleavage o f Pepti d e s 58 M. N- t e r m i n a l Amino A c i d A n a l y s i s by D a n s y l a t i o n Method 58 N. Leucine Amino Pe p t i d a s e (LAP) H y d r o l y s i s o f a Pe p t i d e 60 O. C - t e r m i n a l Amino A c i d D e t e r m i n a t i o n by Carboxypeptidase A 61 P. Amino A c i d A n a l y s i s 62 IV. IDENTIFICATION OF TRYPTIC PEPTIDES ON PEPTIDE MAPS 64 A. Analyses o f Peptides on the Chromatogram of Non AEot A 65 B. Analyses o f A T r y p t i c P e p t i d e Map o f AE(3 c h a i n 74 C. Summary 84 V. Hb j B a n 9 k o k I N A CHINESE NEWBORN IN VANCOUVER, B.C. 85 A. C l i n i c a l S t u d i e s 1. Family study 85 2. He m a t o l o g i c a l i n v e s t i g a t i o n s 85 v i i Page B. B i o c h e m i c a l I n v e s t i g a t i o n 87 1. Hemoglobin s t a r c h g e l e l e c t r o p h o r e s i s and hemoglobin c h a i n d i s s o c i a t i o n study 87 2. I s o l a t i o n o f the abnormal g l o b i n c h a i n 87 3. L o c a t i o n o f abnormal t r y p t i c p e p t i d e on pe p t i d e map 91 4. Amino a c i d a l t e r a t i o n i n TpV o f the v a r i a n t 91 5. L o c a t i o n o f amino a c i d a l t e r a t i o n i n CNBr p e p t i d e , between p o s i t i o n 56-59 o f gTpV 93 6. The s i t e o f mutation i n the 3-chain i s g l y c i n e r e s i d u e 56 which i s r e p l a c e d by a s p a r t i c a c i d 9 7 7. C o n f i r m a t o r y evidence f o r the s i t e o f mutation 100 C. D i s c u s s i o n 100 D. Summary 103 A DOUBLE HETEROZYGOTE OF HbE AND Hb NEW YORK IN A THAI FAMILY 104 A. C l i n i c a l I n v e s t i g a t i o n s 1. G e n t i c study 104 2. Hematological f i n d i n g s 106 B. B i o c h e m i c a l I n v e s t i g a t i o n s 106 1. Hemoglobin e l e c t r o p h o r e s i s 108 2. To demonstrate the mutant g l o b i n c h a i n 108 3. To i s o l a t e both slow and f a s t 3 g l o b i n c h a i n mutants 111 4. To c o n f i r m the i d e n t i t y of the slow 3 v a r i a n t as b e i n g Hb E ( a 2 3 i 6 G 1 u " * L y s ) 113 v i i i Page 5. To l o c a t e the s i t e o f mutation of the f a s t 3-chain v a r i a n t 116 C. The R e l a t i o n o f Amino A c i d A l t e r a t i o n t o Hemoglobin S t r u c t u r e 124 D. D i s c u s s i o n 127 E. Summary 128 V I I . HEMOGLOBIN MAHIDOL - A NEW a-CHAIN MUTANT 130 A. C l i n i c a l , H e m a t o l o g i c a l and G e n e t i c a l Study 130 B. B i o c h e m i c a l I n v e s t i g a t i o n 131 1. Hemoglobin e l e c t r o p h o r e s i s and hemoglobin c h a i n d i s s o c i a t i o n s t u d i e s 134 2. S t r u c t u r a l c h a r a c t e r i z a t i o n o f Hb Mahidol i n S.J. sample 137 3. B i o c h e m i c a l evidence t o show t h a t the f a s t Hb i n S.J. i s a tetramer of 3 g l o b i n c h a i n 157 4. Evidence f o r the i n t e r a c t i o n o f a T h a l a s s -emia and Hb Mahidol 157 5. B i o c h e m i c a l s t u d i e s t o show t h a t S.T. and S.R. are i d e n t i c a l t o Hb Mahidol 159 C. Summary 161 BIBLIOGRAPHY 169 i x LIST OF TABLES Table Page I A summary o f f i n d i n g s r e s u l t i n g from some 43 combinations o f abnormal genes. I I Amino a c i d analyses of t r y p t i c p e p t i d e s from the p e p t i d e map of non AEa A. 71 I I I Amino a c i d analyses o f t r y p t i c p e p t i d e s from the pe p t i d e map o f AE$ A. 79 IV Amino a c i d compositions of methionine p o s i t i v e p e p t i d e (3TpV) of normal and abnormal 8-chain of H b J B a n g k o k . 9 4 V Amino a c i d compositions of CNBr cleavage of 8 ATpV and 8 JTpV. 9 8 VI A summary of h e m a t o l o g i c a l f i n d i n g s and d e s i g n -a t i o n s o f a double heterozygote o f HbE and Hb New York i n a T h a i f a m i l y . 107 E E VII Amino a c i d analyses o f 8 T p I I I - a and 8 TpIII-b p e p t i d e from the p e p t i d e map o f AE8 . 117 A NY V I I I Amino a c i d analyses o f 8 TpXII-b and 8 TpXII-b which were e l u t e d from the p e p t i d e maps o f 5 mg A NY of t r y p t i c p e p t i d e AE8 and AE8 • 120 IX Amino a c i d analyses o f f r e e amino a c i d l i b e r a t i o n NY from the h y d r o l y s i s o f 8 TpXII-b w i t h LAP. 122 X A summary o f h e m a t o l o g i c a l f i n d i n g s o f a-thalassemia Hb Mahidol (S.R.) f a m i l y . 132 XI Amino a c i d analyses o f aTpIX o f normal and v a r i a n t o f Hb Mahidol (S.R. sample). 141 X Table XII Amino acid analyses of the CNBrll-A and CNBrll-i of Hb Mahidol (S.R. sample). XIII Amino acid analyses of the d i l u t e acetic acid cleavage of CNBrII-A peptide. XIV Amino acid analyses of the d i l u t e acetic acid cleavage of CNBrll-M peptide of S.R. sample. XV Amino acid analysis of CNBrll-M of S.T. sample. XVI Amino acid analysis of CNBrll-M of S.R. sample. XVII Amino acid analyses of 0.25 M acetic acid hydrolysis fragments of CNBrll-M of S.T. sample XVIII Amino acid analyses of d i l u t e acetic acid hydrolysis fragments of CNBrll-M of S.R. sample x i LIST OF FIGURES gure Page 1. A diagram o f s t a r c h g e l e l e c t r o p h o r e s i s , t r i s - E D T A -b o r a t e b u f f e r , pH 8.6 shows the m o b i l i t y of HbA 2, HbA3, HbF, Hb Gower-I, Hb Gower-II i n r e l a t i o n t o HbA. 5 2. A diagram o f summarizing the development of g l o b i n c hains i n the human. 8 3. "A diagram showing the conformation and n o t a t i o n o f amino a c i d r e s i d u e s i n h e l i c a l and n o n - h e l i c a l r e g i o n s of sperm whale myoglobin. 10 4. A diagram demonstrating contacts between the heme " group and r e s i d u e s o f the g l o b i n i n a-chain of horse oxyhemoglobin. 13 4a. Contacts between the heme group and r e s i d u e s of g l o b i n i n horse oxyhemoglobin 3-chain. 14 5. A diagram i l l u s t r a t i n g t h i r t y - f o u r r e s i d u e s i n v o l v e d i n hydrogen bonding of o ^ - g 1 c o n t a c t of horse oxyhemoglobin. 16 6. A scheme of n i n e t e e n r e s i d u e s c o n t r i b u t i n g to hydrogen -bonding o f a x - g 2 c o n t a c t o f horse oxyhemoglobin. 17 7. A r e s o l u t i o n o f standard amino a c i d run u s i n g a s i n g l e column system. 63 8. A t r y p t i c p e p t i d e map o f non A E a A w i t h 0.2% . n i n h y d r i n s t a i n . 67 9. A diagram o f the t r y p t i c p e p t i d e map of non AEa wi t h s p e c i a l s t a i n s . 68 x i i F i g u r e Page 9a. A diagram o f the t r y p t i c p e p t i d e map corre s p o n d i n g t o t r y p t i c p e p t i d e number o f AEg on the b a s i s of amino a c i d analyses and s p e c i a l s t a i n s . 68 10. Amino a c i d sequences of t r y p t i c p e p t i d e s o f non AEa c h a i n . 69 A 11. A t r y p t i c p e p t i d e map o f AEg wi t h n i n h y d r i n s t a i n . 76 12. A diagram of the t r y p t i c p e p t i d e map of AEg w i t h s p e c i a l s t a i n s . 77 12a. A p e p t i d e diagram showing the corresponding t r y p t i c p e p t i d e number of AEg- on the b a s i s o f amino a c i d analyses and s p e c i a l s t a i n s . 77 13. Amino a c i d sequences of t r y p t i c p e p t i d e s of AEg . 78 14. The pedigree o f H b J B a n g k o k f a m i l y . 86 15. Hemoglobin s t a r c h g e l e l e c t r o p h o r e s i s , t r i s - E D T A -b o r a t e - b u f f e r , pH 8.6 o f H b J B a n g k o k . 88 16. Hemoglobin c h a i n d i s s o c i a t i o n w i t h PHMB on s t a r c h g e l e l e c t r o p h o r e s i s w i t h t r i s - E D T A - b o r a t e b u f f e r pH 8.6.89 17. C M - c e l l u l o s e chromatography o f H b J B a n g k o k . 90 18. P e p t i d e maps o f t r y p t i c A E g A and A E g J w i t h n i n h y d r i n s t a i n . 92 19. Diagram of the sequence o f g ATpV and g JTpV t r e a t e d w i t h cyanogen bromide. 9 5 20. The paper e l e c t r o p h o r e s i s at pH 6.5 o f cyanogen bromide cleavage p e p t i d e of g ^ p V and g JTpV. 96 x i i i F i g u r e Page 21. T h i n l a y e r chromatography of DNS-amino a c i d s o f A J u n f r a c t i o n a t e d CNBr mixture of g TpV and g TpV. 101 22. Pedigree o f a double h e t e r o z y g o t e of HbE and Hb New York i n a T h a i f a m i l y . ' 105 23. Hemoglobin s t a r c h g e l e l e c t r o p h o r e s i s of a double heterozygote o f HbE and Hb New York. 109 24. S t a r c h g e l e l e c t r o p h o r e s i s i n t r i s - E D T A - b o r a t e b u f f e r pH 8.6 of hemoglobin c h a i n d i s s o c i a t e d by PHMB treatment. 110 25. G l o b i n c h a i n f r a c t i o n a t i o n of the double h e t e r o z y g -ote o f HbE and Hb New York by C M - c e l l u l o s e chromatography. 112 A' E 26. T r y p t i c p e p t i d e maps of AEg and AEg w i t h n i n h y d r i n s t a i n . 114 27. A diagram of known sequence of g ^ p I I I and the amino a c i d a l t e r a t i o n o f the slow g mutant r e s u l t i n g g E T p I I I - a and g E T p I I I - b o b t a i n e d . 115 28. T r y p t i c p e p t i d e maps of A E g A and A E g N Y . 119 29. The sequences o f AEg TpII and the amino a c i d NFY a l t e r a t i o n o f g TpXII-b. 123 30. T h i n l a y e r chromatography o f DNS-amino a c i d s of g ATpXII-b and g N Y T p X I I - b . 125 31. Pedigree of a-thalassemia Hb Mahidol o f S.J. f a m i l y . 1 3 3 32. Hemoglobin s t a r c h g e l e l e c t r o p h o r e s i s o f Hb Mahidol i n t r i s - E D T A - b o r a t e b u f f e r , pH 8.6. 1 35 x i v F i g u r e Page 33. Hemoglobin ch a i n d i s s o c i a t i o n with PHMB treatment on s t a r c h g e l e l e c t r o p h o r e s i s , t r i s - E D T A - b o r a t e b u f f e r . 136 34. C M - c e l l u l o s e chromatography of S.J. g l o b i n . 138 A M 35. T r y p t i c p e p t i d e maps of non AEa and non AEa of Hb Mahidol. 139 36. Paper e l e c t r o p h o r e s i s pH 6.5 o f cyanogen bromide c l e a v e d p e p t i d e o f a^TpIX and a^TpIX. 142 A 37. A diagram of cyanogen bromide cleavage o f a TpIX and A p I X . 14 3 38. P e p t i d e maps o f t r y p s i n and cyanogen bromide cleavage o f non AEot A and non A E a M . 145 . 39. The cadmium n i n h y d r i n s t a i n e d paper e l e c t r o p h o r e s i s pH 1.9 o f guide s t r i p s o f d i l u t e a c e t i c a c i d h y d r o l y s a t e o f C N B r l l - A and CNBrll-M o f S.J. sample. 148. 40. A diagram o f d i l u t e a c e t i c cleavage fragments of C N B r l l - A and CNBrll-M of S.J. sample. 151 41. A t h i n l a y e r chromatography o f DNS-amino a c i d s o f M-4, M-4-a and M-4-b p e p t i d e s . 153 42. The r a t e s o f f r e e amino a c i d r e l e a s e from the h y d r o l y s i s o f M-8 p e p t i d e by carboxypeptidase A. 156 43. C M - c e l l u l o s e chromatography of Hb Mahidol t r a i t . 160 44. Guide s t r i p s o f d i l u t e a c e t i c h y d r o l y s a t e o f CNBrll-M o f S.T. and S.R. sample analyzed by paper e l e c t r o p h -o r e s i s pH 1.9. 162 45. A diagram o f amino a c i d analyses o f d i l u t e a c e t i c c l e a v e d fragments o f CNBrll-M o f S.J., S.T. and S.R. sample. 163 XV ABBREVIATIONS AEa c h a i n = Aminoethylated a g l o b i n c h a i n A a c h a i n = Normal a g l o b i n c h a i n COHb = Carbonmonoxyhemoglobin C M - c e l l u l o s e = Carboxymethyl c e l l u l o s e CNBr = Cyanogen.bromide DNS-amino a c i d = Dansyl amino a c i d Hb = Hemoglobin Hsr = Homoserine H s l = Homoserine l a c t o n e HVE = High v o l t a g e e l e c t r o p h o r e s i s LAP = Leucin e amino p e p t i d a s e PCMB = p-Chloromercuribenzoate PHMB = p-Hydroxymercuribenzoate SAC = S - a m i n o e t h y l c y s t e i n TPCK = L-( l - T o s y l a m i d o - 2 - p h e n y 1 ) e t h y l c h l o r o m e t h y l ketone x v i ACKNOWLEDGEMENTS The author would l i k e t o express h i s g r a t e f u l a p p r e c i a t i o n to P r o f e s s o r G. H. Dixon f o r a d v i c e , guidance and v a l u a b l e d i s c u s s i o n s throughout t h i s i n v e s t i g a t i o n and p r e p a r a t i o n of t h i s t h e s i s . The author a l s o wishes t o acknowledge w i t h g r a t i t u d e the f i n a n c i a l a s s i s t a n c e generously p r o v i d e d by the Canadian I n t e r n a t i o n a l Development Agency, Ottawa. S i n c e r e thanks are a l s o due to Drs. S. Na-Nakorn and P. Wasi, D i v i s i o n o f Hematology, Department of Medicine, Mahidol U n i v e r s i t y , Bangkok, and Dr. G. R. Gray, D i v i s i o n o f Hematology, Department of Pathology, Vancouver General H o s p i t a l , Vancouver, B. C , f o r t h e i r generously s u p p l y i n g the abnormal hemoglobin samples and c l i n i c a l data used i n t h i s study. S p e c i a l thanks are g i v e n t o P r o f e s s o r M. Darrach, Chairman, Department o f B i o c h e m i s t r y , U n i v e r s i t y o f B r i t i s h Columbia f o r h i s encouragement of t h i s study, t o Dr. R. S. Gilmour f o r h i s c o n s t r u c t i v e c r i t i c i s m o f some p a r t s o f the manuscript f o r t h i s t h e s i s , and t o Mr. J . Dergo f o r h i s s k i l l f u l t e c h n i c a l advice i n u s i n g the amino a c i d a n a l y z e r . T h i s work was supported by a grant from the B r i t i s h Columbia M e d i c a l Research Foundation, Vancouver, B. C. x v i i DEDICATION To My Mother 1 INTRODUCTION In 1866 Korber (1) d i s c o v e r e d t h a t the hemoglobin i n the fetus showed more a l k a l i - r e s i s t a n c e than i n the a d u l t . S i n c e t h a t time, two types o f human hemoglobin - a d u l t and f e t a l have been r e c o g n i z e d . Evidence f o r a mutant hemoglobin o c c u r r i n g i n man was foreshadowed i n 1910 when H e r r i c k (2) d e s c r i b e d an anemic p a t i e n t from the West I n d i e s w i t h p e c u l i a r elongated and s i c k l e shaped r e d b l o o d c e l l s . Subsequently the s i c k l i n g phenomenon was observed only a t low oxygen t e n s i o n ( 3 ) . T h i s c l i n i c a l m a n i f e s t a t i o n was l a t e r t o be known as s i c k l e c e l l anemia. J u s t one year b e f o r e the b e g i n n i n g o f the abnormal hemoglobin e r a H o r l e i n and Weber (1948) f i r s t demonstrated t h a t the d e f e c t i n methemoglobinemia occurs i n the g l o b i n p o r t i o n of the molecule w h i l e the heme i s normal ( 4 ) . The avenue to an understanding o f abnormal hemoglobins was opened i n 19 49 when P a u l i n g , Itano, S i n g e r and Wells (5) d i s c o v e r e d t h a t the hemo-g l o b i n i n s i c k l e c e l l anemia p a t i e n t s , d e s i g n a t e d as HbS y possessed a slower m o b i l i t y than a d u l t hemoglobin (HbA) d u r i n g e l e c t r o p h o r e s i s . S i n c e the e l e c t r o p h o r e t i c m o b i l i t y o f a p r o t e i n depends on i t s net charge which i n t u r n i s based on the r e l a t i v e p r o p o r t i o n s of b a s i c and a c i d i c amino a c i d s i n i t s s t r u c t u r e , the term, "molecular d i s e a s e " , f o r s i c k l e c e l l anemia was f i r s t i n t r o d u c e d by P a u l i n g e t a l ( 5 ) . At the same time, two g e n e t i c i s t s - Neel (6) and Beet (7) independently e s t a b l i s h e d t h a t s i c k l e c e l l anemia and s i c k l e c e l l t r a i t r e p r e s e n t e d the homozygous and heterozygous forms r e s p e c t i v e l y of an abnormal hemoglobin gene. 2 S i n c e t h e i n t r o d u c t i o n by P a u l i n g e t a l (5) o f hemoglobin e l e c t r o p h o r e s i s , a l a r g e number o f abnormal hemoglobins have been d e s c r i b e d . However, d i r e c t e v i d e n c e f o r t h e m o l e c u l a r d i s e a s e c o n c e p t was n o t p r o v i d e d u n t i l Ingram (1958) showed by h i s " f i n g e r p r i n t " o r p e p t i d e map t e c h n i q u e t h a t a s i n g l e amino a c i d was a l t e r e d i n HbS - g l u t a m i c a c i d a t t h e 6 t h r e s i d u e o f the 3 - c h a i n was r e p l a c e d by v a l i n e ( 8 ) . T h i s r e s u l t e d i n abnormal e l e c t r o p h o r e t i c b e h a v i o r and i n d i r e c t l y i n t h e c l i n -i c a l m a n i f e s t a t i o n s . As knowledge o f t h e b i o c h e m i s t r y and t h r e e d i m e n s i o n a l s t r u c t u r e o f hemoglobin has advanced i n r e c e n t y e a r s , much l i g h t has been thrown on t h e problem o f h e m o g l o b i n o p a t h i e s and i t has been p o s s i b l e t o c o r r e l a t e b i o c h e m i c a l s t u d i e s w i t h c l i n i c a l and h e m a t o l o g i c a l f i n d i n g s i n terms o f m o l e c u l a r p a t h o l o g y . Thus hemoglobin p r o v i d e s t h e most well-documented example o f t h e s u c c e s s f u l c o r r e l a t i o n o f t h e s t r u c t u r e o f a p r o t e i n w i t h i t s p h y s i o l o g i c a l f u n c t i o n . 3 I . HEMOGLOBIN: STRUCTURE AND GENETICS As i n t e r e s t i n c r e a s e d i n both normal and abnormal hemo-g l o b i n s , s e v e r a l e x c e l l e n t reviews and books have appeared d u r i n g the l a s t few years which p r o v i d e d e t a i l e d i n f o r m a t i o n about v a r i o u s aspects of the s t r u c t u r e and f u n c t i o n o f hemoglobin (9,10,11,12,13,14,15). Human hemoglobin i s composed o f g l o b i n - p o l y p e p t i d e chains and the p r o s t h e t i c group - the i r o n c o n t a i n i n g heme. By the c l a s s i c a l X-ray c r y s t a l l o g r a p h i c s t u d i e s o f P e r u t z , Rossmann, C u l l i s , Muirhead, W i l l and North (16) a hemoglobin molecule has been shown t o c o n s i s t o f fou r p o l y p e p t i d e c h a i n s , each o f which e n f o l d s a s i n g l e heme. F i v e d i f f e r e n t types of g l o b i n chains have been d e s c r i b e d i n human hemoglobin, d e s i g n a t e d as a, 8, v; 6 and e c h a i n . Most hemoglobin molecules c o n s i s t o f two p a i r s o f i d e n t i c a l p o l y p e p t i d e chains w i t h d i f f -e r e n t combinations of a-chain and non a-chain, namely 6, Yf 5 and' e t o form the t e t r a m e r i c s t r u c t u r e s o f HbA, HbF, HbA 2 and embryonic hemoglobin - Gower I I r e s p e c t i v e l y . A. Normal Hemoglobins 1. A d u l t hemoglobins Upon e l e c t r o p h o r e s i s , a t a l k a l i n e pH, a d u l t hemoglobin appears as a heterogenous mixture of hemoglobin f r a c t i o n s , d e s i g n a t e d HbA, HbA 2 and HbA 3 . a) Hemoglobin A - The major hemoglobin component i n a d u l t r e d b l o o d c e l l s , i s prese n t t o the ex t e n t o f 97% o f the t o t a l hemoglobin. Rhinesmith, Schroeder and P a u l i n g (17) 4 showed t h a t the g l o b i n of HbA c o n s i s t s o f one p a i r of a-chains (af) and another p a i r of g - c h a i n s ( g f ) . I t s c h a i n formula i s A A w r i t t e n thus - a2$2. A A? b) Hemoglobin A 2 (01262 ) - A minor hemoglobin, f i r s t d e s c r i b e d by Kunkel, C e p p e l l i n i , M u l l e r - E b e r h a r d and Wolf (18), migrates slower than HbA on e l e c t r o p h o r e s i s a t a l k a l i n e pH ( F i g . 1). I t i s normally p r e s e n t a t a l e v e l 1.5-3% t h a t of the t o t a l hemoglobin i n the a d u l t , w h i l e i n the newborn i t i s p r a c t i c a l l y u n d e t e c t a b l e . The l e v e l o f HbA 2 i s found t o be i n c r e a s e d i n g-thalassemia (18) but decreased i n a-thalassemia (12), 6-thalassemia (19), h e r e d i t a r y p e r s i s t e n c e o f f e t a l hemoglobin (20) and i r o n d e f i c i e n c y anemia (21). c) Hemoglobin A3 - A minor f a s t hemoglobin, d e t e c t e d by e l e c t r o p h o r e s i s a t pH 8.6, runs s l i g h t l y ahead o f HbA toward the anode ( F i g . 1) and i s b e l i e v e d t o be a d e r i v a t i v e of HbA formed d u r i n g i n v i v o ageing of normal red c e l l s (22,23). In a h y b r i d i z a t i o n study, HbA3 d i f f e r e d from HbA i n i t s g-chain (24). S e v e r a l f r a c t i o n s are obtained from HbA3 by IRC 50 chromatography and they probably r e p r e s e n t v a r i o u s secondary HbA d e r i v a t i v e s (25,26). Recent s t u d i e s of one IRC 50 f r a c t i o n showed i t to be a mixed d i s u l f i d e between g l u t a t h i o n e and HbA (27) but a second f r a c t i o n c o n t a i n e d a s u b s t i t u t e d g-chain N terminus (2 8). However, the nature o f HbA3 i s not y e t f u l l y c l e a r . 2. F e t a l hemoglobin (HbF) A F HbF, ( ( X 2 6 2 ) i s the major pigment found d u r i n g f e t a l l i f e . Upon e l e c t r o p h o r e s i s a t pH 8.6, i t migrates j u s t behind 5 Origin HbA 2 HbA HbA 3 Normal adult hemolysate Hb F Hb- Gower I Hb- Gower 1 I Anode F i g u r e 1. A diagram of s t a r c h g e l e l e c t r o p h o r e s i s , t r i s - E D T A -borate b u f f e r , pH 8.6, shows the m o b i l i t y of HbA2, HbA3, HbF, Hb Gower I, Hb Gower I I i n r e l a t i o n t o HbA. 6 HbA ( F i g . 1) and a l s o showed more r e s i s t a n c e t o a l k a l i -d e n a t u r a t i o n than HbA (1). At b i r t h , HbF i s presen t t o the extent of 60-80% o f the t o t a l hemoglobin and r a p i d l y decreases t o the normal low l e v e l a t the end of the f i r s t y e a r . In the a d u l t , i t comprises l e s s than 2% of the t o t a l hemoglobin by the a l k a l i d e n a t u r a t i o n method (29) but i s u n d e t e c t a b l e by e l e c t r o p h o r e s i s . However, i n s e v e r a l c o n d i t i o n s , HbF appears to be i n c r e a s e d , f o r i n s t a n c e , 8-thalassemia (12), s i c k l e c e l l anemia (12), h e r e d i t a r y p e r s i s t e n c e o f f e t a l hemoglobin (20), and v a r i o u s h e m o l y t i c anemias (12,30). 3. Embryonic hemoglobin S t u d i e s o f hemoglobin e l e c t r o p h o r e s i s o f human embryonic hemoglobin (31,32) show t h a t i t c o n s i s t s o f two components, de s i g n a t e d as Hb-Gower I and Hb-Gower I I . a) Hb-Gower I - The f i r s t hemoglobin, i s b e l i e v e d t o be formed i n e a r l y embryonic development a t ^ g e s t a t i o n a l age of about 40 days (33) . At t h a t time only the e-chain i s s y n t h e s i z e d and only the tetramer of e-chain - &•, named Hb-Gower I , i s p r e s e n t . In s t a r c h g e l e l e c t r o p h o r e s i s a t pH 8.6, i t runs s l i g h t l y slower than HbA2 (Figure 1 ) . The amount of Hb-Gower I appears t o decrease when the embryo reaches 40 cm crown-rump l e n g t h and di s a p p e a r s about the t h i r d month of i n t r a u t e r i n e l i f e (32,33). b) Hb-Gower I I - The second embryonic hemoglobin, appears a f t e r the a-chain begins t o be s y n t h e s i z e d and probably c o n s i s t s o f a 2 e-2 (32,34). The m o b i l i t y of t h i s pigment i s 7 slower than Hb-Gower I ( ei») on s t a r c h g e l e l e c t r o p h o r e s i s a t pH 8.6 ( F i g . 1). As the development proceeds, the l e v e l of Hb-Gower I I r a p i d l y f a l l s , presumably because the E-gene i s p r o g r e s s i v e l y i n a c t i v a t e d . No embryonic hemoglobin i s d e t e c t a b l e a f t e r the f e t u s reaches a crown-rump l e n g t h of 100 cm. B. Development o f G l o b i n Chains As mentioned above, the e-chain i s b e l i e v e d to be the f i r s t g l o b i n c h a i n s y n t h e s i z e d d u r i n g e a r l y embryonic develop-ment (33) and i t i s soon f o l l o w e d by a and y c b a i n ( F i g . 2) . However, the e s t r u c t u r a l genes must be completely shut o f f a f t e r the t h i r d month of i n t r a u t e r i n e l i f e . Thus the embryonic hemoglobin - Gower I and Gower II decrease t o und e t e c t a b l e l e v e l s A F and HbF(a2Y2) becomes the major hemoglobin component d u r i n g f e t a l l i f e . The 8-genes b e g i n t o be expressed but are not f u l l y A A f u n c t i o n a l i n the f e t u s so t h a t , HbA(ot2 82 ) i s u s u a l l y p r e s e n t t o an e x t e n t of 20-30% of the t o t a l hemoglobin at b i r t h . However, the 3-genes are f u l l y expressed by 5-6 months and a A F r a p i d i n c r e a s e o f HbA occurs w h i l e the HbF(01272) l e v e l p r o g r e s -s i v e l y d e c l i n e s presumably as a r e s u l t of the y-qenes becoming i n a c t i v a t e d . I t i s a l s o e v i d e n t t h a t t h e r e i s a s w i t c h mechanism c o n t r o l l i n g the r e l a t i v e e x p r e s s i o n o f the 3 and y-genes (35) f o r as y - c h a i n s y n t h e s i s d e c l i n e s , 8-chain s y n t h e s i s i n c r e a s e s and the l e v e l o f t o t a l hemoglobin chains remains r e l a t i v e l y c o n s t a n t . Any circumstances which cause a decrease of 8-chain s y n t h e s i s -e.g. t h a l a s s e m i a major (homozygosity f o r 8-thalassemia) are compensated by p e r s i s t e n c e of y - c h a i n s y n t h e s i s , so t h a t , 8 Figure 2. A diagram summarizing the development of globin chains i n the human. 9 i n s t e a d o f r a p i d l y d e c r e a s i n g a f t e r b i r t h , HbF(012 Y2) w i l l remain as a r e l a t i v e l y l a r g e p r o p o r t i o n o f the t o t a l hemoglobin (29,36). The 6-chain begins t o be s y n t h e s i z e d o n l y j u s t b e f o r e A A? b i r t h so t h a t HbA2(a2<$2 ) i s s c a r c e l y d e t e c t a b l e by s t a r c h g e l e l e c t r o p h o r e s i s i n the newborn. C. Hemoglobin S t r u c t u r e The primary s t r u c t u r e o f human a - g l o b i n c h a i n which c o n t a i n s 141 amino a c i d r e s i d u e s , was e s t a b l i s h e d by H i l l and Konigsberg (37); subsequently the sequences of 3, Y and 6-chains, each o f which co n t a i n s 146 r e s i d u e s were a l s o determined (38,39,40). The e-chain i n embryonic hemoglobin has not y e t been sequenced but p e p t i d e maps showed a p a t t e r n d i f f e r e n t from those o f a, 3/ Y and 6-chain (41). I n t e r e s t i n g l y , the primary s t r u c t u r e of a, 3, Y/ <5 chains and myoglobin (42) as w e l l show a c o n s i d e r a b l e degree o f homology and Ingram (4 3) has suggested t h a t they are a l l d e r i v e d from a common p r e c u r s o r by repeated gene d u p l i c a t i o n s d u r i n g v e r t e b r a t e e v o l u t i o n (44,45). Based on X-ray c r y s t a l l o g r a p h i c s t u d i e s of myoglobin (46) and hemoglobin (16), i t was found t h a t the myoglobin ch a i n A A (150 r e s i d u e s ) as w e l l as a (141 residues) and 3 - c h a i n (146 r e s i d u e s ) share a common t h r e e dimensional conformation although the l e n g t h o f the chains and t h e i r amino a c i d sequences are a p p r e c i a b l y d i f f e r e n t (47). To d e s c r i b e the homologous fe a t u r e s i n conformation the l e t t e r i n g and numbering system proposed by Kendrew e t a l (4 6) i n t h e i r s t u d i e s of the sperm whale myoglobin has been adopted ( F i g . 3). To the e i g h t h e l i c a l 10 F i g u r e 3. A diagram showing the c o n f o r m a t i o n and n o t a t i o n o f amino a c i d r e s i d u e s i n h e l i c a l and n o n - h e l i c a l r e g i o n s o f sperm whale my o g l o b i n (46,47). 11 segments of the g l o b i n c h a i n are a s s i g n e d the l e t t e r s A to H, while the i n t e r h e l i c a l p o r t i o n o r c o r n e r s are denoted by the l e t t e r s o f p r e c e e d i n g and succeeding h e l i c e s . Each amino a c i d r e s i d u e i s numbered from the amino end of each segment, e.g. D7 r e p r e s e n t s the 7th amino a c i d r e s i d u e of h e l i c a l segment D. Segments, NA and HC are r e s p e c t i v e l y the non h e l i c a l amino and carboxy ends o f the c h a i n . In t h i s manner, some p a r t i c u l a r amino a c i d r e s i d u e s become common and meaningful i n terms o f t h e i r c o n t r i b u t i o n to the conformation and s p e c i f i c f u n c t i o n of hemoglobin; f o r example, the heme-linked, proximal h i s t i d i n e i s l o c a t e d a t F8 i n a, 8, and myoglobin and the non-heme-linked, d i s t a l - h i s t i d i n e a l s o occurs a t E7 i n every case. 1. S t a b i l i t y o f the hemoglobin molecule I t i s g e n e r a l l y accepted t h a t the s i d e chains of amino a c i d s can be a s s i g n e d to two l a r g e c l a s s e s . The f i r s t c l a s s possesses h y d r o p h i l i c s i d e chains which r e a d i l y i n t e r a c t w i t h water o r aqueous s o l u t i o n and these are l o c a t e d on the e x t e r i o r o f the hemoglobin molecule. The bonding o f these s i d e chains does not p l a y a s i g n i f i c a n t r o l e i n the s t a b i l i z a t i o n o f the conformation o f the molecule. The second c l a s s possesses hydrophobic s i d e chains which tend to be i n s o l u b l e i n water; i n g e n e r a l they occupy the i n t e r i o r of the hemoglobin molecule which i s , t h e r e f o r e , l a r g e l y hydrophobic (48). I n t e r a c t i o n by hydrophobic bonding, although the f o r c e s per bond are minute, p l a y s a v i t a l r o l e i n the s t a b i l i z a t i o n of the conformation of hemoglobin s i n c e the t o t a l number of i n t e r a c t i o n s i s very 12 l a r g e and the t o t a l bonding energy i s very s i g n i f i c a n t i n m a i n t a i n i n g the g l o b u l a r shape o f each hemoglobin s u b - u n i t . The f o r c e s which m a i n t a i n the conformation of the hemoglobin molecule can be s u b - d i v i d e d as f o l l o w s : a) Hydrophobic bonds - As mentioned above, the hydro-phobic, w a t e r - h a t i n g amino a c i d s occupy the i n s i d e o f the molecule (48) and aggregate themselves t o form i n t e r a c t i o n s known as Van der Waals f o r c e s . In o r d e r f o r these bonds t o be an a p p r e c i a b l e s t a b i l i z i n g f o r c e , water must be t o t a l l y e xcluded from the i n s i d e o f the molecule (4 8). b) Heme b i n d i n g - Each heme l i e s i n a hydrophobic pocket i n the g l o b i n and the i r o n i s c o o r d i n a t e l y l i n k e d to the p r o x i m a l h i s t i d i n e , F8, of each g l o b i n c h a i n . P e r u t z , Muirhead, Cox and Goaman (49) have shown about 60 i n t e r a c t i o n s between atoms which c o n t r i b u t e t o the s t a b i l i z a t i o n o f the heme group i n i t s pocket ( F i g . 4 and 4a). T h i s hydrophobic l i n i n g of the heme pocket i s a b s o l u t e l y e s s e n t i a l t o allow the f e r r o u s i r o n o f the heme i n hemoglobin t o combine r e v e r s i b l y w i t h m o l e c u l a r oxygen. T h i s f u n c t i o n has been p r e d i c t e d by Nakahara and Wang (50) and subsequently e s t a b l i s h e d by Kendrew (51). c) Sub-unit i n t e r a c t i o n s - Although the conformation of g l o b i n c h a i n e s s e n t i a l l y depends on the i n t e r a c t i o n o f the hydrophobic s i d e chains i n s i d e the molecule, f o r complete s t a b i l i t y o f hemoglobin, the i n t e r a c t i o n s between the normal a and non a-chain are a l s o a p p a r e n t l y necessary. F o r example, i t i s known t h a t a f r e e g l o b i n c h a i n w i l l p r e c i p i t a t e r e a d i l y 13 c. a I Figure 4. A diagram demonstrating contacts between the heme group and residues of the globin i n a-chain of horse oxyhemoglobin (49). 14 F i g u r e 4a. Contacts between the heme group and r e s i d u e s of g l o b i n i n horse oxyhemoglobin 3-chain (49). 15 and i n the a-thalassemia syndromes (52,53) where there i s an A F excess of f r e e g and y - c h a i n s , these chains w i l l polymerize A F as tetramers t o form HbH(gi») and Hb B a r t ' s (yt) • These tetramers are known to be e a s i l y denatured and p r e c i p i t a t e d from s o l u t i o n . In the same manner, the f r e e a-chain found i n t h a l a s s e m i a major (homozygosity f o r g-thalassemia) w i l l aggregate t o form i n c l u s i o n s i n the e r y t h r o b l a s t s of bone marrow (54,55). X-ray a n a l y s i s o f HbA (49) has shown t h a t two types of sub u n i t i n t e r a c t i o n s can occur namely u n l i k e c h a i n c o n t a c t s and l i k e c h a i n c o n t a c t s . i . U n l i k e c h a i n i n t e r a c t i o n s - As shown both by the s t u d i e s of Perutz e t a l (49) on the t h r e e dimensional s t r u c t u r e o f HbA and those o f Rosemeyer & Huehns (56) on hemoglobin c h a i n d i s s o c i a t i o n by p-chloromercuribenzoate, c o n t a c t s between a and g-chain can be of two types d e s i g n a t e d as a ^ g 1 (or a 2 - g 2 ) and a*-g 2 (or a 2 - g 1 ) . The a ^ g 1 c o n t a c t r e v e a l s i n t e r a c t i o n s o f 3 4 r e s i d u e s which are mostly hydro-phobic amino a c i d s and about 110 atoms have been shown t o be i n v o l v e d i n t h i s c o n t a c t ( F i g . 5). The a x - g 2 c o n t a c t i s much weaker and i s made up of o n l y 19 r e s i d u e s c o n t r i b u t i n g 80 atoms ( F i g . 6). Because a ^ g 1 c o n t a c t i s s t r o n g e r than a ^ g 2 , the a*-g 2 r e g i o n can separate more e a s i l y than the a ^ g 1 c o n t a c t area d u r i n g the t r a n s i t i o n from oxy- t o deoxyhemoglobin w i t h the r e s u l t t h a t the g-chains move 6°A a p a r t , w h i l e the a-chains move l e s s than 1°A (49). 16 :ln 131 H9 123 H6 126 H9 asp arg 30 B12 Figure 5. A diagram i l l u s t r a t i n g t h i r t y - f o u r residues involved i n hydrogen bonding of a1-^1 contact of horse oxyhemoglobin (49). 17 F i g u r e 6. A scheme o f n i n e t e e n r e s i d u e s c o n t r i b u t i n g t o hydrogen bo n d i n g o f a 1 - 3 2 c o n t a c t o f h o r s e oxyhemoglobin (49). 18 i i . L i k e c h a i n i n t e r a c t i o n s - Although there i s no d i r e c t evidence from the X-ray c r y s t a l l o g r a p h y s t u d i e s , i t i s b e l i e v e d , on the b a s i s o f i n d i r e c t evidence, t h a t there are s a l t b r i d g e s or hydrogen bonds between l i k e c h a i n s ; a-a.and 8~8 c h a i n (49), as f o l l o w s : a-o c h a i n Arg H24 (141) C00~ -- N H 3 + Lys H10 (127) Arg H24 (141) G u a + - i n t e r n a l anion-Gua + Arg H24 (141) Lys G6 (99) N H 3 + - i n t e r n a l a n i o n - N H 3 + Lys G6 (99) 8-8 c h a i n His H24 (146) C00~ — N H 3 + Lys H10 (132) H i s H24 (146) imino-NH 3 + V a l NAl (1) These p o l a r c o n t a c t s probably e x i s t i n d i l u t e b u f f e r s o l u t i o n but are weakened by h i g h s a l t c o n c e n t r a t i o n s . I t i s a l s o e v i d e n t t h a t the 3-8 c o n t a c t must be broken w h i l e the a-a i s only s l i g h t l y a l t e r e d upon deoxygenation (57,58). 2. D i s s o c i a t i o n o f subunits S e v e r a l l i n e s of evidence i n d i c a t e t h a t asymmetrical d i s s o c i a t i o n o f the tetramer o f hemoglobin d i r e c t l y t o momomers; 01282 (tetramer) > 2g+28 (monomer) does not o c c u r . Symmetrical d i s s o c i a t i o n ; 01282 (tetramer) ^—^ 2a8 (dimer) , on the o t h e r hand, occurs r e a d i l y a t h i g h c o n c e n t r a t i o n s of both n e u t r a l e l e c t r o l y t e s and urea or a t a c i d and a l k a l i n e pH 1s (59,60). T h i s symmetrical d i s s o c i a t i o n t o d i m e r i c u n i t s i s r e a d i l y understandable from the X-ray model. I t i s known t h a t h i g h c o n c e n t r a t i o n s o f n e u t r a l e l e c t r o l y t e can weaken p o l a r i n t e r a c t i o n s i . e . the l i k e c h ain c o n t a c t s o f a-a and 8~8, and s t r e n g t h e n the non-polar bonding i . e . the u n l i k e a ^ B 1 c h a i n c o n t a c t s . Thus symmetrical 19 d i s s o c i a t i o n from tetramer t o dimers occurs r e a d i l y f o l l o w i n g the breakage o f the weak, p o l a r bonding between l i k e chains and a*-3 2 c o n t a c t . Although hydrophobic bonds are i n v o l v e d i n the a*~3 2 c o n t a c t t h e i r number i s s m a l l ( 6 1 ) . Very l i t t l e d i s s o c i a t i o n o f dimer i n t o monomer; 2a3 > 2a + 23, can be d e t e c t e d under p h y s i o l o g i c a l c o n d i t i o n s of pH and s a l t c o n c e n t r a t i o n (59) but a p p r e c i a b l e amounts of monomers appear a t pH's below 4.9 o r above 11.0 (6 2 ) . Rosemeyer and Huehns (56) found t h a t an a l t e r n a t i v e means of d i s s o c i a t i n g hemoglobin i s by r e a c t i o n w i t h p-chloromercuribenzoate (PCMB) at an i o n i c s t r e n g t h o f 0.1 and pH 6.0. The PCMB combines f i r s t w i t h the r e a c t i v e s u l h y d r y l group a t c y s t e i n e 9 3 of the 3-chain r e s u l t i n g i n i t i a l l y i n the d i s s o c i a t i o n o f the tetramer to the dimer form, a i 3 i . F u r t h e r r e a c t i o n o f PCMB w i t h the u n r e a c t i v e c y s t e i n e s a t 3-112 and a-104 leads t o complete d i s s o c i a t i o n o f dimers t o monomers as summarized below: r e a c t e d at SH 3-93 a 2 3 2 > 2 a i 3 i tetramer dimers PCMB d i s s o c i a t i o n a t a*-3 2 c o n t a c t 3-112+a-104 2a+23 monomers d i s s o c i a t i o n at a 1 ^ 1 c o n t a c t 20 I I . HEMOGLOBINOPATHIES Hemoglobinopathies, the d i s o r d e r s o f hemoglobin s t r u c t u r e and s y n t h e s i s , are the most wide-spread examples of molecular d i s e a s e . Two major c l a s s e s o f d i s o r d e r are seen, namely, abnormal hemoglobins, and th a l a s s e m i a s . The abnormal hemoglobin i s u s u a l l y the r e s u l t o f a mutation o f one base i n a codon t r i p l e t o f the s t r u c t u r a l gene f o r one of hemoglobin chains which leads t o an amino a c i d s u b s t i t u t i o n i n the primary s t r u c t u r e o f the g l o b i n . Thalassemia apparently m a n i f e s t s i t s e l f as a reduced r a t e of g l o b i n s y n t h e s i s and the most common theory o f i t s cause i s some abnormality o f the r e g u l a t i o n o f g l o b i n s y n t h e s i s . The d i s t r i b u t i o n o f these genes i s w o r l d wide but they occur w i t h h i g h f r e q u e n c i e s i n Mediterranean c o u n t r i e s and South E a s t A s i a . A. Nomenclature o f Abnormal Hemoglobins Since P a u l i n g et a l i n t r o d u c e d hemoglobin e l e c t r o p h o r e s i s (5) as a s c r e e n i n g method, a l a r g e number of v a r i a n t s showing a l t e r e d m o b i l i t i e s have been d e s c r i b e d . In the e a r l y years of abnormal hemoglobin r e s e a r c h new mutants were c a l l e d by the l e t t e r s o f the E n g l i s h alphabet i n the c h r o n o l o g i c a l order o f t h e i r d i s c o v e r y . T h i s nomenclature was i n c o n s i s t e n t i n t h a t the t h r e e hemoglobins; a d u l t , f e t a l and s i c k l e c e l l hemoglobin had a l r e a d y been c a l l e d HbA, HbF and HbS r e s p e c t i v e l y . In the p o i n t o f f a c t , s i c k l e c e l l hemoglobin, the f i r s t mutant should have been c a l l e d HbB but s i n c e i t had a l r e a d y been c a l l e d HbS, the l e t t e r B was omitted and the succeeding new v a r i a n t s were 21 c a l l e d HbC (63), HbD (64), HbE (65) , e t c . Because of the r a p i d l y i n c r e a s i n g number of abnormal hemoglobins and the l i m i t e d number of l e t t e r s i n the alphabet, i t was suggested t h a t HbQ be the l a s t new v a r i a n t named by the l e t t e r i n g system (66). However, new mutants have continued t o be named b e f o r e a meaningful nomenclature was proposed. F o r example, Hb Lepore i s c a l l e d a f t e r the p a t i e n t ' s f a m i l y name (67), Hb B a r t ' s i s an a b b r e v i a t i o n of S t . Bartholomew's H o s p i t a l (6 8), Hb New York i s named a f t e r the c i t y (69). I t i s g e n e r a l l y accepted t h a t a thorough study of the p h y s i c o - c h e m i c a l p r o p e r t i e s o f a new v a r i a n t by comparison wi t h the known abnormal hemoglobins - e.g. i t s b e h a v i o r i n e l e c t r o p h o r e s i s , and the b i o c h e m i c a l c h a r a c t e r i z a t i o n of the mutant i s r e q u i r e d b e f o r e d e s i g n a t i n g a new name f o r the v a r i a n t . For example, many f a s t abnormal hemoglobins showed the same e l e c t r o p h o r e t i c m o b i l i t y as HbJ which was f i r s t d e s c r i b e d by Thorup, Itano, Wheby and L e a v e l l (70) but the amino a c i d a l t e r a t i o n s were q u i t e d i f f e r e n t , thus the c o u p l i n g of HbJ w i t h d e s c r i p t i v e names was proposed; as i n H b J B a l t i m o r e ( a A 0 ] e A s p ) ( 7 1 ) , H b J T o r o n t o ( a | A s p 3 A ) ( 7 2 ) > H b J B a n g k o k 6 A s P ) ( ? 3 / ? 4 ) ^ T h e f a c t t h a t a l l known abnormal hemoglobins are not a v a i l a b l e f o r a comparison of e l e c t r o p h o r e t i c m o b i l i t i e s makes i t e s s e n t i a l t h a t complete b i o c h e m i c a l c h a r a c t e r i z a t i o n be c a r r i e d out b e f o r e a new hemoglobin mutant i s d e s c r i b e d . 22 B * Abnormal Hemoglobins Over one hundred abnormal v a r i a n t s have now been d e s c r i b e d (15,75,76). The c l a s s i f i c a t i o n and l i s t s are the f o l l o w i n g : 1. H e r e d i t a r y abnormal hemoglobins a. Amino a c i d s u b s t i t u t i o n i n p o l y p e p t i d e c h a i n i . S i n g l e amino a c i d s u b s t i t u t i o n a) Alpha Chain Hemoglobin Residue H e l i c a l From To Reference NO. No. J Toronto 5 A3 A l a Asp Nature 208:1059 (1965) J P a r i s 12 A10 A l a Asp A c t a Haemat. 39 : 291 (1968) J Oxford 15 A13 Gly Asp Nature 204: 269 (1964) I I n t e r l a k e n 15 A13 Gly Asp A c t a Haemat.(Jap) 32: 9 (1964) I 16 A14 Lys Glu Nature 207: 259 (1965) I Texas 16 A14 Lys Glu Nature 214: 499 (1967) J M e d e l l i n 22 B3 Gly Asp Fed. Proc. 23: 172 (1964) Memphis 23 B4 Glu Gin J . Lab. C l i n . Med.66 : 886 (1965 23 B4 Glu Gin Biochem. 5: 3701 (1966) G Aud h a l i 23 B4 Glu V a l Nature 219: 1164 (1968) G Honolulu 30 B l l Glu Gin B i o l . Chem. 237: 1517 (1962) G Singapore ff ft t i 11 IT P t l f! t l G Hong Kong i t 11 t l ir If 17 11 t t »" T o r i n o 43 CD1 Phe V a l Nature 217: 1016 (1968) Umi 47 CD6 Asp Gly / Kokura ••• i f / L F e r r a r a 47 CD6 Asp Gly Proc.Med.(Naples) 16: 553 (1960 Sealy 47 CD6 Asp His Amer.J.Hum.Genet. 20: 151 (1968 Hasharon 47 CD6 Asp His I s r a e l J.Med.Sci. 3: 827 (1967) / C i t e d by Sc h r o e d e r , W n a t u r a l P rod. 23: 113 .A. and (1965) J o n e s , R. T., P r o g . Chem. O r g a n i c 23 J Sardegna 50 CD8 His Russ 51 CD9 Gly Shimonoseki 54 E3 Gin Mexico 54 E3 Gin N o r f o l k 57 E6 Gly G l b ad an 57 E6 Gly L P e r s i a n G u l f 57 E6 Gly M Boston 58 E7 His M Osaka 58 E7 His G P h i l a d e l p h i a 68 E l 7 Asn G B r i s t o l 68 E17 Asn G S t 1 68 E17 Asn D Alpha S t . Louis 68 E17 Asn X 68 E17 Asn S t a n l e y v i l l e 1 68 E17 Asn Ube 2 68 E17 Asn Mahidol 74 EF3 Asp S t a n l e y v i l l e 2 78 EF7 Asn Ann Arbor 80 F l Leu Etobicoke 84 F5 Ser G No r f o l k 85 F6 Asp M Kankakee 87 F8 His M S h i b a t a 87 F8 His t C i t e d by Lehmann , I i . , and C a r r e l l Asp Nature 218: 470 (1968) Arg Biochim.Biophys.Acta 130: 541 (1966) Arg Biochim.Biophys.Res.Commun. 11: 229 (1963) Blood 24: 624 (1964) Glu C l i n . Res. 11: 105 (1961) Asp B i o l . Chem. 237: 69 (1962) Asp Brit.J.Haemat. 6: 140 (1960) Arg A c t a Haemat. 42: 169 (1969) Tyr Proc.Nat.Acad.Sci.(U.S.) 47: 1758 (1958) Tyr Biochim.Biophys.Acta 97: 472 (1965) Lys Biochim.Biophys.Acta 48: 253 (1961) Lys Biochim.Biophys.Acta 86: 144 (1964) Lys Nature 211: 1305 (1966) Lys / Lys Nature 189: 465 (1961) Lys / Asp Clin.Chim.Acta 16: 347 (1967) His Can. J . Biochem. (19 70) Lys B r i t . Med. J . 4; 92 (1968) Arg t Arg Can. J . Biochem. 47: 143 (1969) Asn t T y r J . B i o l . Chem. 241: 2137 (1966) Tyr Acta.Haematol.(Jap)26: 531 (196 , R.U., B r i t . Med. B u l l . 25. 14 (1969 24 M Iwate 87 F8 His Broussais 90 FG2 Lys Ches apeake 92 FG4 Arg J Cape Town 92 FG4 Arg Manitoba 10 2 G9 Ser Dakar 112 G19 His Chiapas 114 GH2 Pro J T o n g a r i k i 115 GH3 A l a 0 Indonesia 116 GH4 Gl u Bibb a 136 GH19 Leu D Alpha TpIV Q TpIX B e i l i n s o n TpVI b) Beta Chain Tokuchi 2 NA2 His S 6 A3 Glu C 6 A3 Glu C Georgetown 7 A4 Glu G 7 A4 Glu S i r i r a j 7 A4 Glu Porto Alegre 9 A6 Ser Sogn 14 A l l Leu T y r Acta.Haematol.(Jap)26: 538 (19* Biochim.Biophys.Acta 107: 226 (1965) Asn C P , Soc. B i o l . 160; 2270 (19GG Leu J . M o l . B i o l . 19: 91 (1966) Gin Nature 212: 792 (1966) Arg Crookston, G o l d s t e i n , Lehmann and Beale (Unpublished) Gin t Arg Biochim.Biophys.Acta 154: 488 (1968) Asp J.Med.Genetics 4: 1 (1967) Lys Nature 207: 259 (1965) Pro Biochim.Biophys.Acta 154: 220 (1968) Nature 182: 852 (1958) H i l l and Jones Brit.J.Haemat. 9 : 484 (1963) T y r B u l l . Yamaguchi Med. Sch. 10 : 1 (1963) V a l Nature 180: 326 (1957) Lys Nature 184: 640 (1959) Lys New.Eng.J.Med. 268: 862 (1963) Gly J.Biol.Chem.235: 3182 (1960) Lys B r i t . M e d . J . 1: 1583 (1965) Cys S c i e n c e 158: 800 (1967) Arg Scand.J.Haematol, ( i n press) 25 D Bushman 16 A13 Gly J B a l t i m o r e 16 A13 Gly J T r i n i d a d 16 A13 Gly J I r e l a n d 16 A13 Gly J New Haven 16 A13 Gly E Saskatoon 22 B4 Glu G Saskatoon 22 B4 Glu G Coushatta 22 B4 Glu F r e i b u r g 23 B5 V a l G Taiwan-Ami 25 B7 Gly E 26 B8 Glu Genova 28 BIO Leu Tacoma 30 B12 Arg P h i l l y 35 C l T y r Hammersmith 42 CD1 Phe G Galveston 43 CD2 Glu G Texas 43 CD 2 Gl u G P o r t A r t h u r 43 CD2 Gl u K Ibadan 46 CD5 Gly G Copenhagen 47 CD6 Asp J Bangkok 56 D7 Gly J Meinung 56 D7 Gly J Korat 56 D7 Gly Arg Nature 216: 688 (1967) Asp Biochim.Biophys.Acta 78: 637 (1963) Asp / Asp / Asp Biochim.Biophys.Res. Commun. 16: 368 (1964) Lys Canada J.Biochem. 45: 1385 (196 A l a Canada J.Biochem. 45: 351 (196"i A l a Biochem.Biophys. Res. Commun. 26: 466 (1967) Science 154: 1024 (1967) Arg B i o c h e m . B i o p h y s . R e s . Commun. 30 : 690 (1968) Lys Biochim.Biophys .Acta 49 : 520 (1961) Pro Nature 214: 877 (1967) Ser t Phe t Ser Nature 216: 663 (1967) A l a Blood 23: 193 (1964) A l a B l o o d 2 3 : 193 (1964) A l a B l o o d 2 3 : 193 (1964) Glu Nature 208: 658 (1965) Asn Biochim.Biophys.Acta 140: 231 (1967) Asp B r i t . J . H a e m a t o l . 13: 303 (1967) J . M o l . B i o l . 19: 91 (1966) Asp Biochim.Biophys.Res.Commun. 24: 732 (1966) Asp I s r a e l J . Med. S c i . 1. 768 (196 l i i k a r i 61 E5 L y s i\i S e a t t l e 61 E5 L y s M S a s k a t o o n 63 E7 H i s M Emory 63 E7 H i s I-l Kurume 63 E7 H i s i i C h i c a g o 63 E7 H i s K Hamburg 63 E7 H i s Z u r i c h 63 E7 H i s l i M i l w a u k e e 1 67 E l l V a l S y d n e y 67 E l l V a l J C a m b r i d g e 69 E13 G l y J Rambam 69 o r 83 E13 G l y K o r l e Bu 73 E17 A s p S e a t t l e 76 E20 A l a J I r a n 77 EF1 H i s G A c c r a 79 E F 3 A s p D I b a d a n 87 F3 T h r S a n t a Ana 88 F4 L e u A g e n o g i 90 F6 G l u S a b i n e 91 F7 L e u M hyde P a r ] ; 92 F8 H i s Oak R i d g e 94 F G l A s p N Memphis 95 FG2 L y s A s n C l i n . C h i m . A c t a 10. 101 (1964) G l u B i o c h i m . B i o p h y s . A c t a 154- 278 (1968) T y r P r o c . N a t . A c a d . S c i . ( U . S . ) 47: 1758 (1961) T y r P r o c . N a t . A c a d . S c i . ( U . S . ) 47; 1758 (1961) T y r A c t a . H a e m a t o l . ( J a p ) 25; 6 9 0 ( 1 9 6 T y r J . L a b . C l i n . M e d . 59. 918 (1962) T y r P h y s i o l . C h e m i e . 345: 181 (19 66) A r g B i o c h i m . B i o p h y s . A c t a 5 0 : 5 9 5 ( 1 9 6 G l u P r o c . W a t . A c a d . S c i . ( U . S . ) 47: 1758 (1961) A l a N a t u r e 215: 626 (19 67) A s p B i o c h i m . B i o p h y s . A c t a 140. 231 ( 1 9 6 7 ) ' A s p I s r a e l J . M e d . S c i . 1: 836 (1965 A s n J . M e d . G e n e t . 5: 107 (1968) G l u f A s p B r i t . M e d . J . 1: 674 (1967) A s n N a t u r e 2 0 3 ; 363 (1964) L y s N a t u r e 212: 792 (1966) P r o J . M e d . G e n e t . 5: 257 (1968) L y s C l i n . C h i n . A c t a 14. 624 (1966) P r o New E n g . J . Med. (19 69) T y r P r o c . 1 1 t h C o n g . I n t . S o c . H e m a t o l . 427 (1966) A s n t G l u o r G i n / 27 Hopkin 1 95 FG2 Lys N Jenkins 95 FG2 Lys N Baltimore 95 FG2 Lys Koln 98 FG5 V a l Kempsey 99 FG6 Asp Yakima 99 FG6 Asp Kansas 102 G4 Asn New York 113 G15 V a l Hi jiyama 120 GH3 Lys D Los Angeles 121 GH4 G l u D Punjab 121 GH4 Glu D Cyprus 121 GH4 Glu D Conley 121 GH4 Glu D Chicago 121 GH4 Glu D P o r t u g a l 121 GH4 Glu 0 A r a b i a 121 GH4 G l u Hofu 126 H4 V a l K Cameroon 129 H7 A l a Wien 130 H8 Tyr K Woolwich 132 H10 Lys Hope 136 H14 Gly Kenwood 143 H21 His R a i n i e r 145 HC2 Tyr G l u Nature 214. 189 (1967) Glu Biochim.Biophys.Acta 117: 492 (1966) Glu Nature 207: 945 (1965) Met Nature 210: 915 (1966) Asn Blood 31: 623 (1968) His J . C l i n . I n v e s t . 46: 1840 (1967) Thr Fed.Proc. 26: 673 (1967) G l u Nature 213: 876 (1967) Glu S c i e n c e 159: 204 (1968) Gin Biochim.Biophys.Acta 86: 163 (1964) Gin Biochim.Biophys.Acta 59: 437 (1962) Gin Gin Gin Gin Lys Nature 196: 229 (1962) Glu Nature 217: 89 (1968) Glu or Asp Nature 208: 658 (1965) Asp Pietschmann e t a l (unpublished) Gin Nature 208: 658 (1965) Asp Blood 25: 830 (1965) Asp Nature 207: 259 (1965) His Science 159: 741 (1968) 28 F Texas 1 F Texas 2 F Alexandra F H u l l *F x A 2 Sphakia A 2 N.Y.U. A 2 or B 2 A 2 F l a t b u s h A 2 Babinga a) Gamma Chain 5 A2 6 A3 12 A9 121 GH4 136 d) D e l t a Chain 2 NA2 12 A9 16 A13 22 B4 136 H14 Glu Lys B r i t . J . H a e m a t o l . 13: 252 (1967) G l u Lys B r i t . J . H a e m a t o l . (196 8) Thr Lys t Glu Lys B r i t . M e d . J . 3: 531 (1967) Gly A l a Proc.N.A.S. 60: 537 (1968) His Arg J.Biol.Chem. 241: 2137 (1966) Asn Lys I s r a e l J . Med. S c i . 3: 327 (196 Gly Arg Nature 209: 1217 (1966) A l a G l u Sc i e n c e 151: 1406 (1966) Gly Asp Nature 219: 1360 (1968) i i . Two amino a c i d s u b s t i t u t i o n s i n p o l y p e p t i d e Hb C Harlem a 2 A 3 6 g l u -*Val, 73 A s p A s n J.Biol.Chem. 242 :248 (1967) b. P o s s i b l e abnormality d u r i n g meiosis Hemoglobin Abnormal Chain P o s s i b l e Mechanism Hb Lepore(Boston) Non-alpha c h a i n c o n s i s t s Unequal c r o s s i n g over with of N-terminal d e l t a f u s i o n o f 6-3 genes, c h a i n (6Tpl-5) and C- t e r m i n a l b e t a c h a i n (6Tpl2-15) Reference - Biochem.Biophys.Acta 97: 37 (1965) Non-alpha c h a i n c o n s i s t s Unequal c r o s s i n g over w i t h o f N - t e r m i n a l d e l t a c h a i n f u s i o n o f 6-3 genes. (6Tpl-3) and C - t e r m i n a l b e t a c h a i n (3Tp5-15) Reference - Proc.Nat.Acad.Sci. (U.S.) 48: 1880 (1962) 91-95 o f 3-chain absent Unequal c r o s s i n g over w i t h d e l e t i o n o f n u c l e o t i d e s of f i v e amino a c i d s . Hb Lepore (Hollandia) Hb Gun H i l l Reference - Science 157: 1581 (1967) *Found i n some cases of normal c o r d b l o o d . 29 Hb F r e i b u r g Hb Leiden Lepore V a r i e t y Bos ton Cyprus H o i l a n d i a Augus t a Pylos The Bronx V a l of 23rd amino a c i d Unequal c r o s s i n g over w i t h r e s i d u e of $-chain d e l e t i o n o f n u c l e o t i d e s of absent v a l . amino a c i d . Reference: Science 154: 1024 (1966) G l u o f 6th or 7th r e s i d u e of 3-chain d e l e t e d Reference: Nature 220: 788 (1968) Reference Proc.Nat.Acad.Sci.(U.S.) 48: 1880 (1962) Brit.J.Haemat. 10: 159 (1964) Nature 194: 931 (1962) Biochim.Biophys.Acta 127: 428 (1966) Proc.Nat.Acad.Sci.(U.S.) 48: 1880 (1962) Nature 204: 163 (1964) c. Chain aggregations due to alpha c h a i n d e f i c i e n c y Hemoglobin Hb H Hb Ba r t ' s Hb 6 A 2 Formula F Y«. .A2 o it Hb Gower I Reference J.Amer.Chem.Soc. 181: 3161 (1959) Nature 184: 872 (1959) Biochem.Biophys.Res.Commun. 7: 444 (1962) Nature 201: 1095 (1964) d. Other abnormal hemoglobin Hb P o r t l a n d y 2 X 2 Science 157: 65 (1967) 2. A c q u i r e d abnormal hemoglobins Abnormal Mechanism Hemoglobin Hb K o e l l i k e r C - t e r m i n a l end o f a-chain the Arg. 141 b e i n g m i s s i n g . Reference: A c t a Haemat. 37: 174 (1967) The a c t i o n o f carboxy-peptidase i n the serum. F a s t Hb a s s o c i a t e d S i m i l a r t o Hb A w i t h Pb, tolbutamide P o s s i b l e complex of GSSG w i t h g A2 Reference: Blood 28: 377 (1966) 30 Hb H i n Leukemia ? ? A c q u i r e d Reference: B r i t . J . Haematol, 6: 171 (1960) Nature 199: 1297 (1963) C. How the s t r u c t u r e of abnormal hemoglobins r e l a t e s t o t h e i r  m o l e c u l a r pathology. Although i t has been p o s s i b l e f o r a number of years t o l o c a t e the p r e c i s e p o i n t of mutation i n the primary s t r u c t u r e o f abnormal hemoglobins, i n many cases the r e l a t i o n between the b i o c h e m i c a l l e s i o n s and the h e m a t o l o g i c a l and c l i n i c a l m a n i f e s t a t i o n s i s s t i l l obscured, In other words, a number of p a t i e n t s showing any of s e v e r a l abnormal hemoglobins i n the heterozygous or even the homozygous forms appear to be normal. In c o n t r a s t , o t h e r mutations even i n the h e t e r o z y g o u s e s t a t e , cause h e m a t o l o g i c a l and c l i n i c a l d i s o r d e r s . An e x p l a n a t i o n of these o b s e r v a t i o n s comes from the s t u d i e s of Perutz and h i s co-workers (49) who have e s t a b l i s h e d the d e t a i l s o f the three dimensional s t r u c t u r e and atomic i n t e r a c t i o n s of the hemoglobin molecule as d e s c r i b e d b r i e f l y i n the p r e v i o u s s e c t i o n . Based on t h i s d e t a i l e d knowledge of hemoglobin s t r u c t u r e , i t i s c l e a r t h a t o n l y c e r t a i n areas o f the molecule p l a y an e s s e n t i a l r o l e i n the s t a b i l i z a t i o n o f the s t r u c t u r e and t h a t any a l t e r a t i o n i n these s e n s i t i v e areas w i l l l e a d t o c o n f o r m a t i o n a l changes i n hemoglobin and l i k e l y c l i n i c a l consequences. At the p r e s e n t time, i t i s p o s s i b l e to r a t i o n a l i z e the c l i n i c a l e f f e c t s o f many mutants i n terms of the known th r e e d i m e n s i o n a l s t r u c t u r e s of hemoglobin but i n a few cases, a complete c o r r e l a t i o n between the s t r u c t u r a l a l t e r a t i o n and c l i n i c a l f i n d i n g s has not been p o s s i b l e 31 A c c o r d i n g to the mol e c u l a r s t r u c t u r e o f hemoglobin, i t i s p o s s i b l e to c l a s s i f y the hemoglobin replacements as f o l l o w s : 1. Amino a c i d a l t e r a t i o n i n the r e g i o n o f the heme group As a l r e a d y mentioned, the heme-globin c o n t a c t area i s composed o f two p a r t s ; the i r o n atom - -co-ordinate l i n k a g e a t the proximal h i s t i d i n e F8 and the oth e r c o - o r d i n a t e i n t e r -a c t i o n s o f i r o n with the d i s t a l h i s t i d i n e E7, and the p y r r o l e r i n g n i t r o g e n s . F u r t h e r s t a b i l i z a t i o n occurs by the e x t e n s i v e non-polar c o n t a c t s of the hydrophobic groups on heme w i t h the hydrophobic amino a c i d s surrounding a t the heme pocket. Mutation can occur i n any o f these key heme-globin c o n t a c t areas. a. Replacement o f h i s t i d i n e - Both proximal and d i s t a l h i s t i d i n e s are e s s e n t i a l f o r m a i n t a i n i n g the i r o n i n the f e r r o u s s t a t e , t h i s , i n t u r n , being e s s e n t i a l f o r r e v e r s i b l e combination w i t h m o l e c u l a r oxygen. Amino a c i d a l t e r a t i o n s a t t h i s s i t e , f o r example, i n Hb M Iwate - the proximal h i s t i d i n e replacement by t y r o s i n e , a f 7 T y r 3 A , the phenolate group of t y r o s i n e can l i n k t o and s t a b i l i z e the i r o n as an i r r e v e r s i b l e f e r r i c s a l t r e s u l t i n g i n severe methemoglobinemia and cyanosis ( 7 7 ) . S e v e r a l examples of t h i s type o f mutation, known c o l l e c t i v e l y as Hb M, have been r e p o r t e d , namely: Proximal h i s t i d i n e F8 - a-chain - Hb M I w a t e • a f 7 T y r B A 3-chain - Hb M Hyde Park a A 3 2 2 T y r D i s t a l h i s t i d i n e E7 - a-chain - Hb M Boston a 2 8 T y r 8 A 3-chain - Hb M Saskatoon a A 3 t 3 T y r 32 I t i s i n t e r e s t i n g to study the pathology o f Hb M Milwaukee and Hb Z u r i c h i n terms o f the molecular s t r u c t u r e and hence reach an understanding of the c l i n i c a l d i s o r d e r s . Hb M Milwaukee ( a A B 2 ?Va-'-">G-'-u) produces methemoglobinemia j u s t l i k e the oth e r Hb M mutants although the mutation occurs a t n e i t h e r the proximal nor the d i s t a l h i s t i d i n e (78). However, i t i s p o s s i b l e to understand the d i s o r d e r by examining the molecular i n t e r a c t i o n s based on the three dimensional hemoglobin s t r u c t u r e . V a l i n e 867 ( E l l ) i s l o c a t e d very c l o s e t o the d i s t a l h i s t i d i n e (E7) by v i r t u e o f being the 4th r e s i d u e from i t i n an a - h e l i c a l s t r u c t u r e and when i t i s r e p l a c e d by glutamic a c i d i n Hb M Milwaukee, the "o'-glutamyl c a r b o x y l group can form a s a l t b r i d g e with i r o n thus s t a b i l i z i n g i t i n the f e r r i c s t a t e and pushing the d i s t a l h i s t i d i n e out o f the heme pocket ( 7 7 ) . In Hb Z u r i c h ( a A 8 l 3 H l s " * " A r 9 j d e s p i t e the replacement o f the d i s t a l h i s t i d i n e (E7) by a r g i n i n e , no methemoglobinemia or cyanosis occurs ( 7 9 ) . T h i s i m p l i e s t h a t the i r o n i s s t i l l a b le to r e v e r s i b l y combine with m o l e c u l a r oxygen. As an e x p l a n a t i o n , i t has been proposed t h a t a r g i n i n e i s too b i g to f i t i n t o the heme pocket and must protrude o u t s i d e , l e a v i n g a l a r g e space a t the l i g a n d s i t e of i r o n atom ( 7 7 ) . T h i s s u b s t i t -u t i o n leads t o a d e s t a b i l i z a t i o n o f the heme group and i s c o n s i s t e n t with the c l i n i c a l f e a t u r e s o f a more e a s i l y o x i d i z e d and p r e c i p i t a t e d hemoglobin which leads to i n c l u s i o n body anemia (80) . 33 b. R e p l a c e r a e n t o f n o n - p o l a r c o n t a c t s s u r r o u n d i n g t h e heme. The n o n - p o l a r c o n t a c t s b e t w e e n t h e heme g r o u p a n d n o n - p o l a r a mino a c i d s i d e c h a i n s l i n i n g t h e heme p o c k e t a r e e s s e n t i a l f o r t h e t i g h t b i n d i n g o f heme a n d a l s o f o r t h e r e v e r s i b l e c o m b i n a t i o n o f f e r r o u s i r o n w i t h m o l e c u l a r o x y g e n ( 4 9 , 5 1 ) . Any amino a c i d s u b s t i t u t i o n f r o m a n o n - p o l a r t o a p o l a r r e s i d u e w o u l d d i s r u p t t h e Van d e r W a a l s b o n d i n g a n d l e a d t o t h e r e l e a s e o f t h e heme; t h i s , i n t u r n r e s u l t i n g i n g l o b i n c h a i n d i s s o c i a t i o n and p r e c i p i t a t i o n . T h e s e c h a n g e s a t t h e m o l e c u l a r l e v e l a r e c o n s i s t e n t w i t h c l i n i c a l d i s o r d e r s o f H e i n z b o d y a n e m i a and m e t h e m o g l o b i n e m i a i n some c a s e s . A t l e a s t 7 v a r i a n t s h a v e b e e n d e s c r i b e d a t t h e p r e s e n t t i m e ; V a r i a n t R e p l a c e m e n t H e l i c a l No. C l i n i c a l M a n i f e s t a t i o n db T o r i n o <t 3 P h e + V a l 0 A a 2 32 CD1 I n c l u s i o n b o d y a n e m i a lib B i b b a i 3 6Leu-*-Pro QA U2 p 2 1119 I n c l u s i o n b o d y a n e m i a hb Hammersmith AD i+ 2Phe-*-Ser a 2 3 2 CD1 I n c l u s i o n b o dy a n e m i a Hb S y d n e y A Q 6 7Val->Ala a 2 3 2 E l l I n c l u s i o n b o d y a n e m i a l i b S a n t a Ana A „ 8 s L e u + P r o 0t 2 U 2 F4 I n c l u s i o n body a n e m i a Hb K o l n A D 9 8 V a l + M e t a 2 3 l FG5 I n c l u s i o n b o d y a n e m i a lib K a n s a s A„ i o 2 Asn-VThr a 2 3 2 G4 C y a n o s i s a n d u n s t a b l e Hb K a n s a s , t h e s u b s t i t u t i o n a t A s n 3102 (G4) b y T h r n o t o n l y p r e s e n t s t h e c l o s e c o n t a c t w i t h t h e v i n y l and t h e m e t h y l g r o u p o f p y r r o l e I I w h i c h l e a d s t o heme i n s t a b i l i t y , b u t i s a l s o i n v o l v e d i n t h e a J - 3 2 c o n t a c t ( 7 0 , 8 1 ) ( s e e t h e r e p l a c e m e n t a t a*-3 2 c o n t a c t ) . 34 2. Amino a c i d a l t e r a t i o n s a t s u b u n i t c o n t a c t s As p r e v i o u s l y mentioned, two types o f su b u n i t c o n t a c t s ; between l i k e chains and u n l i k e c h a i n s , occur i n the hemoglobin molecule. The c o n t a c t s between l i k e chains are p o l a r (49, 61) and these area r e a d i l y i n t e r a c t w i t h s o l v e n t w i t h o u t c a u s i n g any c o n f o r m a t i o n a l change. T h e r e f o r e amino a c i d s u b s t i t u t i o n s a t these c o n t a c t s would not be expected to a l t e r the s t a b i l i t y o f the hemoglobin molecule; as an example, i n Hb K Woolwich ( a A B l 3 2 L y s " * * G l u ) , the replacement o f l y s i n e H10 by gl u t a m i c a c i d which o c c u r s i n the p o l a r 8-8 c o n t a c t area i s appa r e n t l y w ithout e f f e c t s i n c e both l y s i n e and glu t a m i c a c i d are h i g h l y p o l a r (77). In c o n t r a s t , the c o n t a c t s between u n l i k e sub u n i t s , b e i n g most non-polar i n nature, are s e n s i t i v e t o amino a c i d a l t e r a t i o n as f o l l o w s : a. Amino a c i d replacement a t the a 1 - 8 2 c o n t a c t - The a x - 8 2 c o n t a c t appears t o be e s s e n t i a l f o r r e s p i r a t o r y f u n c t i o n and i n i t i a t i o n o f tetramer d i s s o c i a t i o n t o dimers (49,61). In the t r a n s i t i o n from oxy- to deoxyhemoglobin, the a 1 - 8 2 c o n t a c t moves and there i s a r o t a t i o n a p a r t o f the g-chains by about 6°A, the a - c h a i n s , however, move l e s s than 1°A (16,49). T h i s c o n f o r m a t i o n a l change upon oxygenation i s b e l i e v e d t o be i n v o l v e d i n the heme-heme i n t e r a c t i o n . Rosemeyer and Huehns (56) a l s o demonstrated t h a t tetramer d i s s o c i a t i o n t o dimer (a8) o c c u r r e d at the a 1 - 8 2 c o n t a c t . Thus amino a c i d s u b s t i t u t i o n s a t t h i s s i t e would be expected t o show a l t e r a t i o n s i n r e s p i r a t o r y f u n c t i o n and tetramer d i s s o c i a t i o n . Most of the mutations at 35 t h i s s i t e appear to i n c r e a s e oxygen a f f i n i t y , and t o d i m i n i s h heme-heme i n t e r a c t i o n and p a t i e n t s p o s s e s s i n g these abnormal hemoglobins show the c l i n i c a l f e a t u r e s of polycythemia as consequence. In Hb Kansas, the only mutant o f t h i s group whose s u b - u n i t d i s s o c i a t i o n has been s t u d i e d shows i n c r e a s e d d i s s o c i a t i o n i n t o a g dimers (77,81). Other mutants i n a ^ g 2 c o n t a c t area are as f o l l o w s : C l i n i c a l M a n i f e s t a t i o n Cyanos i s ^Polycythemia Cyanosis Cyanosis Cyanos i s Cyanosis b. Amino a c i d replacements a t the a ^ g 1 c o n t a c t - The a x - g x c o n t a c t , which c o n t a i n s mostly non-polar bonds, i s known as the i n t e r s u b - u n i t s t a b i l i z e r between a and g - c h a i n s forming the dimer of a g . Any mutation at t h i s s i t e would i n c r e a s e the r a t e of dimer d i s s o c i a t i o n i n t o monomers. However, d i s s o c i a t i o n t o monomers g e n e r a l l y does not occur under p h y s i o l o g i c a l c o n d i t i o n s but only a t low o r high pH (62). T h e r e f o r e , an amino a c i d replacement p a r t i c u l a r l y i n the heterozygote would not be expected t o show any abnormality under p h y s i o l o g i c a l c o n d i t i o n s . In f a c t , c o n s i s t e n t v/ith t h i s p r e d i c t i o n , the heterozygotes o f t h i s c l a s s of mutation do mostly appear normal. Mutations i n t h i s group are the f o l l o w i n g : V a r i a n t Replacement H e l i c a l No. Hb J Capetown 9 2Arg-*-Gln RA « 2 P 2 FG4 Hb Chesapeake « 2Arg-*Leu f tA 0.2 P 2 FG4 Hb Yakima R 9 9Asp-*-His a 2 P 2 FG6 Hb Kemps ey R99Asp->-Asn 0-2 P2 FG6 Hb Kansas ftl02Asn+Thr a 2 P 2 G4 V a r i a n t Replacement H e l i c a l No. C l i n i c a l M a n i f e s t a t i o n Hb G Chinese n 3 o Glu-*Gln f tA 0-2 P 2 B l l None Hb Chiapas 0 | i » P r o + A r g B A GH2 None Hb E 0 i B | , G l u * L y 8 B8 None Hb Tacoma n ft3oArg-*-Ser & 2 P 2 B12 Increased oxygen a f f i n i t y Hb P h i l l y „ o 3 -Fyr+Phe » 2 P 2 C l M i l d h e m o l y t i c anemia Hb New York „ a 11 3Val->-Glu C*2 P 2 G15 None 3. Amino a c i d a l t e r a t i o n s a t i n t e r n a l p o s i t i o n s i n the hemoglobin molecule. As has been c l e a r l y e s t a b l i s h e d , the i n t e r i o r o f the hemoglobin molecule i s occupied almost e x c l u s i v e l y by hydro-phobic r e s i d u e s and t h e i r i n t e r a c t i o n s i n t h i s non-polar m i l i e u are an important source o f s t a b i l i t y o f the g l o b u l a r s t r u c t u r e . Thus, amino a c i d a l t e r a t i o n i n s i d e the molecule may cause v a r y i n g degrees o f c o n f o r m a t i o n a l change dependent on the type of a l t e r a t i o n . For i n s t a n c e , the d i s r u p t i o n o f g l o b u l a r s t r u c t u r e due t o d e l e t i o n o f amino a c i d s , such as i n Hb F r e i b u r g (82), Hb Gun H i l l (83) and Hb Le i d e n (84), o r to replacement i n Hb Genova of l e u c i n e by p r o l i n e which i s i n c o m p a t i b l e w i t h an a - h e l i c a l s t r u c t u r e , leads to d e n a t u r a t i o n and p r e c i p i t a t i o n o f hemoglobin which i s seen as i n c l u s i o n body h e m o l y t i c anemia (85). However, the amino a c i d s u b s t i t u t i o n i n some cases, e.g. Hb Et o b i c o k e (86), Hb Hope (87), Hb Manitoba (88), appear c l i n i c a l l y normal but the p h y s i c o - c h e m i c a l s t u d i e s show abnormal p r o p e r t i e s , such as heat l a b i l i t y , and sometimes g r e a t d i f f i c u l t y i n s e p a r a t i n g the mutant from HbA i n s p i t e 37 of a d e f i n i t e charge change as a r e s u l t o f the amino a c i d replacement (77). The mutants t h a t f a l l i n t o t h i s group are l i s t e d below: V a r i a n t Replacement H e l i c a l No. D i s o r d e r s Hb F r e i b u r g a A 8 l 3 V a l " > d e l e t « B 5 Cyanosis, high oxygen a f f i n i t y Hb Gun H i l l a A g i 1 ~ 9 7 d e l e t * F7-FG4 Hemolytic anemia, absence of heme i n g-chain Hb Leiden A Q 6 o r 7 G l u - d e l e t (X2 P 2 X "A3 o r A4 Hb Wien a A 3 x 3 0 T y r - A s p H8 Hemolytic anemia, u n s t a b l e Hb Hb Genova a A g i s L e U " P r o BIO Hemolytic anemia, heat l a b i l e Hb Hope a A 3 i 3 6 G l y - A s p H14 D i f f i c u l t t o separate from Hbi Hb Sogn A Q i i^Leu+Arg A l l I n creased r a t e of d e n a t u r a t i o i Hb Manitoba lozSer+Arg A Ct2 P 2 G9 D i f f i c u l t to separate from Hbi Hb Etobicoke e^Ser-»-ArgRA <*2 P2 F5 Heat l a b i l e Hb 4. Amino a c i d a l t e r a t i o n s on the o u t s i d e o f hemoglobin molecule. The m a j o r i t y of amino a c i d s u b s t i t u t i o n s i n abnormal hemoglobins occur on the s u r f a c e of hemoglobin molecule where the s t a b i l i t y i s not a f f e c t e d , t h e r e f o r e , the hemoglobin and c l i n i c a l f i n d i n g s o f t e n appear normal i n h e t e r o z y g o t e s . However, the homozygosity f o r some mutants such as Hb C present as c l i n i c a l d i s o r d e r s (14) but the mo l e c u l a r pathogenesis i s s t i l l obscure. d e l e t * = d e l e t i o n • r e s i d u e 91-95 or 92-96 o r 93-97 are d e l e t e d 38 D. Thalassemias In 19 25 Cooley and Lee f i r s t d e s c r i b e d , i n f i v e c h i l d r e n , a new c l i n i c a l syndrome i n v o l v i n g h e m o l y t i c anemia, hyper-pigmentation of s k i n , bone changes, growth r e t a r d a t i o n and abnormal red c e l l morphology (89). Subsequently, t h i s c l i n i c a l d i s o r d e r was known as Cooley's anemia. However, another name, th a l a s s e m i a , has a l s o been i n t r o d u c e d by Whipple and B r a d f o r d (90) i n o r d e r to emphasize the h i g h i n c i d e n c e of the d i s e a s e i n the Mediterranean r a c e s . Knowledge of the g e n e t i c i n h e r i t a n c e of the c o n d i t i o n commenced when Wintrobe, Mathews, P o l l a c k and Dobyns (91) f i r s t made the o b s e r v a t i o n t h a t the parents and some s i b s o f Cooley's anemia p a t i e n t s showed a m i l d degree of red c e l l a b n o rmality. V a l e n t i n e and Neel (9 2) e s t a b l i s h e d t h a t the Cooley's anemia and the c a r r i e r c o n d i t i o n r e p r e s e n t e d the homozygous and heterozygous s t a t e s r e s p e c t i v e l y of a d e f e c t i v e gene. T h i s concept was confirmed by e x t e n s i v e f u r t h e r s t u d i e s (93,94). At the p r e s e n t , i t i s r e a l i z e d t h a t t h i s group of abnormal genes shows not o n l y a w o r l d wide spread but a l s o c o n s i d e r a b l e h e t e r o g e n e i t y . 1. D e s i g n a t i o n o f Thalassemias Thalassemia d e f i n e d as h e r e d i t a r y d i s o r d e r with a reduced r a t e of g l o b i n c h a i n s y n t h e s i s and presents as a hypochromic m i c r o c y t i c anemia without i r o n d e f i c i e n c y . The d i a g n o s i s of a t h a l a s s e m i a i s made on the b a s i s o f three c r i t e r i a , namely, h e m a t o l o g i c a l , b i o c h e m i c a l and g e n e t i c a l evidence. 39 a. Hematological evidence - A hypochromic m i c r o c y t i c anemia w i t h excess i r o n i n t i s s u e s i s the c h a r a c t e r i s t i c h e m a t o l o g i c a l f e a t u r e . T h i s c o n d i t i o n can be r e c o g n i z e d by hypochromia, m i c r o c y t o s i s , a n i s o c y t o s i s , p o i k i l o c y t o s i s and t a r g e t c e l l s on the p e r i p h e r a l b l o o d smear. Furthermore the abnormal morphology of red c e l l s i s a l s o s u b s t a n t i a t e d by d e c r e a s i n g red c e l l i n d i c e s and osmotic f r a g i l i t y . The presence of i r o n such as i n bone marrow, r e t i c u l o - e n d o t h e l i a l t i s s u e or a t l e a s t normal serum i r o n i s r e q u i r e d to exclude i r o n d e f i c i e n c y anemia which can a l s o l e a d t o such red c e l l a b n o r m a l i t i e s . b. B i o c h e m i c a l evidence - The s u g g e s t i o n t h a t t h a l a s s e m i a might be due to a reduced r a t e o f g l o b i n s y n t h e s i s was f i r s t made by Itano (95) . T h i s i d e a has now been c l e a r l y s u b s t a n t i a t e d by s t u d y i n g hemoglobin s y n t h e s i s i n r e t i c u l o c y t e s of t h a l a s s e m i c p a t i e n t s i n v i t r o (96,97,98). From evidence o f the s u g g e s t i o n of a from 8 gene l o c i and s i n c e the major a d u l t hemoglobin con t a i n s both a and 3,-chains, Ingram and S t r e t t o n (99) have proposed two major t y p e s : a and g-thalassemia. A p a t i e n t with. B-thalassemia showed decreased 8-chain s y n t h e s i s , but the y and 6 c h a i n s y n t h e s i s appears to be somewhat i n c r e a s e d r e s u l t i n g i n the r e d u c t i o n o f Hb A c o n c e n t r a t i o n while Hb F and A 2 are e l e v a t e d (12,99,100). In a-thalassemia where there i s a reduced r a t e of a-chain s y n t h e s i s , the minor hemoglobins; Hb F and Hb A 2, would not be expected t o be i n c r e a s e d but there i s a r e l a t i v e excess of f r e e y and 3-chains which can polymerize to form r e s p e c t i v e l y yX-Hb B a r t ' s , and 8A-Hb H (12,99,100). 40 c. G e n e t i c evidence - S t u d i e s o f f a m i l y pedigreedare e s s e n t i a l f o r d e t e c t i n g the s e g r e g a t i o n o f abnormal t h a l a s s e m i a genes. T h i s i s e s p e c i a l l y t rue i n the case o f combination o f genes, e.g. i n a p a t i e n t showing double h e t e r o z y g o s i t y o f both a t h a l a s s e m i a and an abnormal hemoglobin o r d i f f e r e n t types o f th a l a s s e m i a s . 2 . a-Th a 1 ass emi as In the newborn, a - t h a l a s s e m i a i s not d i f f i c u l t t o F recognxze on the basxs o f the presence o f Hb B a r t ' s (y^) o r A Hb H (3i») . W e a t h e r a l l (101) has demonstrated a s m a l l amount of Hb B a r t ' s even i n the normal newborn cord b l o o d by s t a r c h g e l e l e c t r o p h o r e s i s but. always a t l e s s than 1%; t h i s has been a s c r i b e d t o a very s l i g h t imbalance o f g l o b i n c h a i n s y n t h e s i s i n f e t a l l i f e . In c o n t r a s t , the a-thalassemia t r a i t i n the a d u l t i s h a r d l y d e t e c t a b l e e i t h e r by h e m a t o l o g i c a l o r b i o c h e m i c a l s t u d i e s ( 1 2 , 1 0 0 ) . From the i n h e r i t a n c e o f Hb H d i s e a s e e s t a b l i s h e d by Wasi, Nar-Nakorn and Suingdumrong (102) i t i s c l e a r t h a t a t l e a s t two sub-types o f ot-thalassemias e x i s t , namely a - t h a l a s s e m i a i ( a - T h a l i ) - the c l a s s i c a l a-thalassemia and a - t h a l a s s e m i a 2 ( a - T h a l 2 ) - a m i l d a - t h a l a s s e m i a . a. a - T h a l a s s e m i a i ( c l a s s i c a l a-thalassemia) - A c c o r d i n g t o P o o t r a k u l , Wasi and Na-Nakorn (103), the heterozygous s t a t e i n the newborn w i l l p r e s e n t 5-6% of Hb B a r t ' s withered Id degree of t h a l a s s e m i c r e d c e l l changes. T h i s f a s t pigment disa p p e a r s i n the f i r s t few months of l i f e but the abnormal r e d c e l l s w i l l p e r s i s t w i t h normal o r low Hb A 2 and Hb F i n t o the l a t e r c h i l d h o o d and a d u l t l i f e . 41 b. a - t h a l a s s e m i a 2 ( m i l d a-thalassemia) - I t i s p o s s i b l e t o diagnose t h i s genotype i n newborns but extremely d i f f i c u l t to r e c o g n i z e i n the a d u l t except on the b a s i s of g e n e t i c s t u d i e s (102). From o b s e r v a t i o n s of the c o r d b l o o d of 30 newborn o f f s p r i n g o f Hb H parents (10 3a) i t was c l e a r t h a t a - T h a l 2 cases show 1-2% o f Hb Bar t ' s w i t h very m i l d r e d c e l l changes i n the newborn. Furthermore, s t a t i s t i c a l evidence from subsequent s t u d i e s of 1408 newborns i n Bangkok (104) a l s o confirmed t h i s concept. 3. 3-Thalassemias In c o n t r a s t to a-thalassemia, 6-thalassemia i s r a t h e r d i f f i c u l t t o r e c o g n i z e i n i n f a n t s because the 3 l o c i are not f u l l y f u n c t i o n a l . However, a f t e r one year o f age, when the 8 l o c i s h ould be completely f u n c t i o n a l , the h e m a t o l o g i c a l and b i o c h e m i c a l a b n o r m a l i t i e s w i l l be pronounced. I t i s g e n e r a l l y accepted t h a t at l e a s t two types of 8-thalassemias occur, namely A 2 - t h a l a s s e m i a and F - t h a l a s s e m i a (86-thalassemia) (11,12,100,104). a. A 2 - t h a l a s s e m i a - In t h i s c o n d i t i o n , e l e v a t i o n o f Hb A 2 w i t h a s l i g h t l y i n c r e a s e d Hb F i s c h a r a c t e r i s t i c of heterozygous s t a t e . From a study of the combination o f A 2 - t h a l a s s e m i a w i t h Hb E i n T h a i l a n d (104), the A 2 - t h a l a s s e m i a can be d i v i d e d i n t o two sub-types, c a l l e d c l a s s i c a l A 2 - t h a l a s s e m i a and m i l d A 2 - t h a l a s s e m i a on the b a s i s of i n t e r -a c t i o n with Hb E. The former shows severe s u p p r e s s i o n of A. 8 s y n t h e s i s r e s u l t i n g i n the absence o f d e t e c t a b l e Hb A, 42 w h i l e the l a t t e r shows onl y p a r t i a l d e p r e s s i o n o f 8 s y n t h e s i s l e a d i n g to the appearance of some Hb A upon s t a r c h g e l e l e c t r o p h o r e s i s . Furthermore, the l e v e l s o f Hb A 2 t h a t occur i n two sub-types ( i d e n t i f i e d g e n e t i c a l l y ) were s t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t (104). b. F - t h a l a s s e m i a - T h i s c o n d i t i o n i s s i m i l a r t o the A 2 - t h a l a s s e m i a except t h a t t h e r e i s decreased Hb A 2 but e l e v a t e d Hb F. Only the presence o f Hb F without Hb A and Hb A 2 has been d e s c r i b e d i n homozygous s t a t e (105,106). E. Combinations of Abnormal Genes As i s g e n e r a l l y accepted, hemoglobinopathies are manifested i n two c l i n i c a l forms: abnormal hemoglobins and t h a l a s s e m i a s . The former c o n s i s t o f two major groups - a and 8 mutants and the l a t t e r are a l s o d i v i d e d i n t o two main types, d e s i g n a t e d as a and 8-thalassemias. Furthermore, each type of t h a l a s s e m i a can be sub-typed as d e s c r i b e d above. Thus, t h e r e are a number of p o s s i b i l i t i e s f o r d i f f e r e n t combinations of these abnormal genes to occur l e a d i n g to a v a r i e t y o f c l i n i c a l syndromes ranging from c o n d i t i o n s t h a t are asymptomatic to those incompatible with l i f e . Some combinations have a l r e a d y been w e l l r e c o g n i z e d . Table 1, i s a summary o f the c h a r a c t e r i s t i c of h e m a t o l o g i c a l , b i o c h e m i c a l and c l i n i c a l f i n d i n g s i n which the abnormal genes p r e s e n t were a s c e r t a i n e d g e n e t i c a l l y , a c c o r d i n g t o P o o t r a k u l , Wasi and Na-Nakorn (103,107,108). T a b l e I - A Summary of F i n d i n g s R e s u l t i n g From Some Combinations o f Abnormal Genes* Newborn C h i l d o r A d u l t Type Combinations Genotype Hb type RBC Hb type RBC C l i n i c a l F i n d i n g s C l i n i c a l Designatior a Thal x+ HbBarts 90% ++++ otThali t r a c e A+F S t i l l b i r t h o r dead s h o r t l y a f t e r b i r t h Hb B a r t ' s hydrops f e t a l i s Homozygote 3Thal + 3Thal A+F A+F C+A 2 r h a l ++++ Apparently h e a l t h y a t b i r t h but prog-r e s s i v e l y develops d i s e a s e i n i n f a n c y Thalassemia major aThalx+ oiThal 2 A+F+Barts Hb Bart's 25-30% c i n c l u s i o n +++ A.+H ++++ c i n c l u s i o n Double heterozygote Normal appearance a t b i r t h b u t Hb H d i s e a s e d i s e a s e develops i n f i r s t few months of age ctThal + 3Thal A+F+Barts Bart's 1-6% A + A2 s l i g h t l y i n c r e a s e d Healthy as heterozygote Double heterozygote o f a and 3 T h a l a s s -emia based on gene-t i c evidences o n l y Homozygote E+E E ( A 2 t ) + F T a r g e t E only c e l l s T a r g e t c e l l s A p p arently h e a l t h y Pure E Abnormal Hbs Double heterozygote E+NY not r e p o r t e d expeo- E+NY but expected t e d E(A 2+)+F+ normal New York Appar-e n t l y normal Healthy Double heterozygote of HbE and Hb NY conti n u e d . . . *The f i n d i n g s are based on the s t u d i e s on h e t e r o g e n e i t y o f over 2500 newborns i n Bangkok, T h a i l a n d by P o o t r a k u l , Wasi and Na-Nakorn (108), some p a r t s have a l r e a d y been d e s c r i b e d (10 3,104,107) and the ot h e r s w i l l be p u b l i s h e d . T a b l e I (continued) Newborn C h i l d or Adult Type Combinations Genotype Hb type RBC Hb type RBC C l i n i c a l F i n d i n g s C l i n i c a l DesignatiLor otThal + ctchain mutant aThali+Hb Not d e s c r i - Not des-_Mahidol+H ++++ Mahidol bed but cribed c i n c l u s -expected but ex- ion body Hb Mahidol+ pected slow HbF+ ++ Hb Bart's Disease form i n adult aTha1assemia-Hb Mahidol disease Thai and abnormal Hbs 6Thal+ gchain mutant BThal + HbE E ( A 2 + )+F ++ E+F ++++ Apparently normal gThalassemia-at b i r t h b ut pro- HbE d i s e a s e g r e s s i v e l y d e v e l -ops d i s e a s e i n 1st few months o f age aTh a l i + HbE E ( A 2 + )+F+A+ B a r f s 1-2% + A+E EVL7-25% + Healthy as E t r a i t otThalassemiai HbE otThal 1 + HbE E(A 2t)+F+A B a r t ' s 1-2% + o r + A+E E^17-25% + o r + Healthy as E t r a i t otThalassemia 2 HbE otThal and HbE otThal 1 + otThal 2 + HbE E(A 2t)+F+A ++ Ba r t ' s 25-30% few i n c l u s . E+A+Bart's c few i n c l u s . ++++ C l i n i c a l f i n d i n g s AE Bar t ' s d i s e a s e are s i m i l a r t o HbH d i s e a s e otThal i+ E(A 2t)+F+ homozygote B a r t ' s of HbE 5-6% + and Target c e l l s HbE o n l y + and Tar g e t c e l l s L i k e pure E otThalassemiai and pure E aThal 2 + E(A 2t)+F+ homozygote B a r t ' s o f HbE 1-2% + p r + STarget c e l l s HbE on l y + o r + STarget c e l l s S i m i l a r t o pure E aThala s s e m i a 2 and pure E Note: The degree of thalassemic red c e l l changes are graded i n + to ++++, representing mild to severe abnormality, while + i s very mild changes. •fa. 45 I t i s e v i d e n t t h a t the severe i n t e r a c t i o n of genes does not occur i n combinations between d i f f e r e n t types of genes at d i f f e r e n t l o c i , such as, a-thalassemia and 3-thalassemia (104,109) or a-thalassemia and g-chain mutant - a-thalassemia Hb E (104,110) but the i n t e r a c t i o n does e x i s t i n combinations of the same type o f genes which are a l l e l i c o r c l o s e l y l i n k e d or a t the same l o c i (12,100,104). For i n s t a n c e , the combination of two a-thalassemia genes, the homozygote of a - T h a l j , i s known as Hb B a r t ' s hydrops f a t a l i s (107,111,112,113); double h e t e r o z y g o s i t y f o r a - T h a l i and a - T h a l 2 m a n i f e s t s as Hb H d i s e a s e (104); between 0-thalassemias, the homozygous s t a t e r e s u l t s i n the c l i n i c a l syndrome of t h a l a s s e m i a major (89,9 2); between a-thalassemia and a mutant - a - t h a l a s s e m i a Hb Mahidol d i s e a s e and between 3-thalassemia and 3 mutant as 3-thalassemia Hb E d i s e a s e (104). The i n t e r a c t i o n of genes r e s u l t s i n a c l i n i c a l syndrome w i t h severe c l i n i c a l , h e m a t o l o g i c a l and b i o c h e m i c a l a b n o r m a l i t i e s . The c l i n i c a l f i n d i n g s are w e l l r e c o g n i z e d and i n c l u d e a severe h e m o l y t i c anemia, j a u n d i c e , hepatosplenomegaly, bone changes on X-ray and growth r e t a r d a t i o n . A severe degree o f morpholog-i c a l abnormality of the red c e l l s w i t h i n c r e a s e d t i s s u e i r o n s t o r e s i s an obvious f i n d i n g . B i o c h e m i c a l i n t e r a c t i o n s are a l s o shown i n which the severe s u p p r e s s i o n of normal g l o b i n c h a i n s y n t h e s i s r e s u l t s i n almost complete absence of Hb A i n the p a t i e n t . 46 I I I . MATERIALS AND METHODS A. Heme-lys ate P r e p a r a t i o n Blood samples were from two sources; the f i r s t , by c o u r t e s y o f Dr. G. R. Gray from the D i v i s i o n o f Hematology, Department of Pathology, Vancouver General H o s p i t a l , B. C , the second from Drs. Na-Nakorn and Wasi of the D i v i s i o n o f Hematology, Department o f Medicine, S i r i r a j M e d i c a l S c h o o l , Mahidol U n i v e r s i t y , Bangkok. Venous b l o o d , w i t h EDTA as a n t i c o a g u l a n t , was c e n t r i f u g e d t o remove plasma, the r e d c e l l sediment was washed th r e e times w i t h 0.9% NaCl. In the case o f samples from T h a i l a n d , the washing process was performed i n Bangkok and packed red c e l l s were f r o z e n i n d r y - i c e f o r t r a n s p o r t a t i o n to Vancouver. A f t e r thawing, the r e d c e l l s were agai n l y s e d w i t h two volumes of water and 0.5 volumes of t o l u e n e . The mixture was v i g o r o u s l y shaken and kept over n i g h t i n the c o l d room (4° C ) . The toluene l a y e r on the top was removed and the c l e a r hemolysate was o b t a i n e d by f i l t e r i n g r and c e n t a f u g i n g a t 20,000 r.p.m. f o r 20 minutes. For s t o r a g e , carbonmonoxy hemoglobin was prepared by s a t u r a t i o n of the hemoglobin s o l u t i o n w i t h carbonmonoxide o b t a i n e d by adding s m a l l volumes o f 90% f o r m i c a c i d s o l u t i o n t o c o n c e n t r a t e d s u l f u r i c a c i d a t 60° C. T h i s procedure was done i n the fume hood w i t h c a u t i o n . B. S t a r c h Gel E l e c t r o p h o r e s i s H o r i z o n t a l s t a r c h g e l e l e c t r o p h o r e s i s was c a r r i e d out w i t h t r i s - E D T A - b o r a t e b u f f e r , pH 8.6 a c c o r d i n g to Smithies (114) 4 7 w i t h s l i g h t m o d i f i c a t i o n s f o r economy and b e t t e r r e s o l u t i o n (115) . T h i r t y grams of h y d r o l y s e d s t a r c h (Connaught, Toronto) was suspended i n 320 ml. o f g e l b u f f e r a f t e r cooking w e l l by s w i r l i n g i n a b o i l i n g water bath and degassing, the s t a r c h s o l u t i o n was poured i n t o a L u c i t e t r a y w i t h i n n e r dimensions 23x12x0.6 cm. The s u r f a c e of the g e l was l e v e l e d by g e n t l y r a i s i n g or l o wering the margin o f the t r a y . A l i d w i t h t h i n (0.5 mm) s l o t formers was p l a c e d on top o f the molten g e l b e i n g c a r e f u l to exclude a i r bubbles and was f i x e d a t each end with m o d e l l i n g c l a y . The g e l was ready f o r use a f t e r s t a n d i n g f o r 30 minutes at room temperature and 30 minutes i n the c o l d room. The l i d was then removed. The hemolysate, approximately 6 gm% c o n c e n t r a t i o n , was a p p l i e d i n t o the s l o t s u s i n g the thumb and middle f i n g e r to open them. Excess sample, s o l u t i o n was removed with a P a steur p i p e t t e . The h o r i z o n t a l g e l was run a t 270-300 v o l t s f o r 10-12 hours i n the c o l d room. A f t e r running f o r 30 minutes, when the hemoglobin s o l u t i o n had moved away from the o r i g i n i n t o the g e l , the s u r f a c e was covered w i t h Saran wrap t o prevent the g e l from d r y i n g out. The g e l was s l i c e d i n t o two p i e c e s . The top l a y e r was s t a i n e d with Amido b l a c k 10B i n a c e t i c a c i d and the bottom w i t h b e n z i d i n e s t a i n (0.1 gm of b e n z i d i n e was d i s s o l v e d i n 5 ml of g l a c i a l a c e t i c a c i d and water added t o make 10 0 ml. J u s t b e f o r e s t a i n i n g , 0.2 ml of 30% H 2 O 2 was mixed.). 48 C. Hemoglobin Chain D i s s o c i a t i o n With p-Hydroxymercuribenzoate (PHMB) Hemoglobin c h a i n d i s s o c i a t i o n by p-mercuribenzoate was f i r s t d e s c r i b e d by B u c c i and F r o n t i c e l l i (116). T h i s compound i s known to b l o c k the f r e e s u l f h y d r y l group o f c y s t e i n y l r e s i d u e s o f a and B-chairs and t h i s m o d i f i c a t i o n leads t o sub-u n i t d i s s o c i a t i o n . The r e a c t i o n i s as f o l l o w s : Prot-SH + Ho-Hg-^O^-cooNa P r o t - S - H g - ^ O j - c o o N a + H 20 g l o b i n c h a i n PHMB As p r e v i o u s l y d e s c r i b e d , the s u l f h y d r y l group a t 393 r e a c t s f i r s t w i t h PHMB and t h i s d i s r u p t s the o^-B 2 c o n t a c t w i t h d i s s o c i a t i o n o f the tetramer i n t o dimer s u b - u n i t s . F u r t h e r r e a c t i o n o f PHMB wi t h the SH group o f 8112 and al04 takes p l a c e and the a 1 - 8 1 i n t e r s u b - u n i t c o n t a c t i s broken and f i n a l l y the dimers d i s s o c i a t e i n t o monomers. Carbonmonoxy hemoglobin, a t about 1 gm%, was r e a c t e d w i t h excess PHMB (Sigma Chemical Co. L o t 120B-641-10) i n 0.2 M NaCl and phosphate b u f f e r w i t h i o n i c s t r e n g t h 0.1, a t pH 6, a c c o r d i n g to Rosemeyer and Huehns (56) . For convenience, the r e a c t i o n mixture and c o n t r o l were s e t up as f o l l o w s : Phosphate B u f f e r COHb NaCl Water PHMB 1=0.2 10gm% 2M C o n t r o l (unreacted) 1 ml 0.2 ml 0.2 ml 0.6 ml 0 Reacted hemoglobin 1 ml 0.2 ml 0 .2 ml 0.6 ml 2 mg The hemoglobin mixture was covered w i t h p a r a f i l m and kept at 4° C f o r 36 hours. The supernatant o f hemoglobin s o l u t i o n was a p p l i e d t o a s t a r c h g e l w i t h t r i s - E D T A - b o r a t e b u f f e r i n 49 the presence o f PHMB (10 mg/L was added t o the g e l b u f f e r and e l e c t r o d e v e s s e l s o l u t i o n ) . D. G l o b i n P r e p a r a t i o n G l o b i n was prepared from an hemolysate by p r e c i p i t a t i o n i n 20 volumes excess o f 2% HCl-acetone a t -20° C. The super-natant, c o n t a i n i n g heme, was decanted. The p r e c i p i t a t e d g l o b i n was washed w i t h c o l d acetone and sedimented f o u r times a t low speed i n order t o remove t r a c e s o f a c i d and heme. F i n a l l y the p r e c i p i t a t e was d r i e d under n i t r o g e n gas and a g r a y i s h - w h i t e powder o b t a i n e d . E. G l o b i n Chain S e p a r a t i o n The g l o b i n chains were sep a r a t e d by C M - c e l l u l o s e chromat-ography i n sodium phosphate b u f f e r , c o n t a i n i n g 8M urea and 0.05M 8-mercaptoethanol, at pH 6.7, a c c o r d i n g t o Clegg , Naughton and W e a t h e r a l l (74). 8M ure a s o l u t i o n was prepared f r e s h l y b e f o r e the experiment by d i s s o l v i n g the s o l i d urea i n water and warming up i n water bat h a t 40-50° C. I n s o l u b l e i m p u r i t i e s were removed by f i l t r a t i o n and the c l e a r s o l u t i o n was then d e i o n i z e d i n a 3x50 cm column o f mixed-bed r e s i n (AG 501-x8, 20-50 mesh, Bio-Rad) which had been prewashed w i t h d i s t i l l e d water. The d e i o n i z e d urea was c o l l e c t e d a f t e r d i s c a r d i n g the f i r s t 150 ml o f e f f l u e n t . I f the c o n d u c t i v i t y o f urea was over 10 umftbs, the r e s i n was renewed. A s t a r t i n g b u f f e r c o n t a i n i n g 0.05M 3-mercaptoethanol and 0.005M Na2HPOi» i n 8M urea was prepared and the pH was 50 a d j u s t e d to 6.7 w i t h phosphoric a c i d . The con c e n t r a t e d b u f f e r was made i n the same manner as the s t a r t i n g b u f f e r except t h a t the m o l a r i t y o f sodium phosphate was 0.03M. The c o n d u c t i v i t y o f the s t a r t i n g and the con c e n t r a t e d b u f f e r was approximately 0.45-0.5 and 2.1-2.2 m i l l i mohs r e s p e c t i v e l y . E i t h e r new or regenerated C M - c e l l u l o s e (Cellex-CM, Bio-Rad) was suspended i n the s t a r t i n g b u f f e r and a d j u s t e d t o pH 6.7 w i t h IN NaOH. The u n s e t t l e d f i n e s a f t e r 15-20 minutes s t a n d i n g were decanted and the s e t t l e d C M - c e l l u l o s e was twice re-suspended i n s t a r t i n g b u f f e r . The s e t t l e d C M - c e l l u l o s e was f i n a l l y packed i n the column under the pre s s u r e from a column p a c k i n g rubber b u l b u n t i l the C M - c e l l u l o s e bed was over 18 cm i n h e i g h t . The packed column was washed w i t h the s t a r t i n g b u f f e r u n t i l the o p t i c a l d e n s i t y and c o n d u c t i v i t y o f the e f f l u e n t were s a t i s f a c t o r y . G l o b i n was d i s s o l v e d i n the s t a r t i n g b u f f e r a t a concent-r a t i o n o f 20 mg/ml and d i a l y z e d a g a i n s t two changes of 50 volumes of s t a r t i n g b u f f e r f o r a p e r i o d o f th r e e hours a t the room temperature. The d i a l y z e d sample was then a p p l i e d t o the CM - c e l l u l o s e column, which was then washed w i t h s t a r t i n g b u f f e r i n o r d e r t o remove unbound m a t e r i a l s u n t i l the absorbance and the c o n d u c t i v i t y o f the e f f l u e n t r e t u r n e d t o t h a t o f the s t a r t i n g b u f f e r . G l o b i n chains were then e l u a t e d a t room temperature w i t h a l i n e a r N a + g r a d i e n t o b t a i n e d by mixing equal volumes of the s t a r t i n g and co n c e n t r a t e d b u f f e r . The e f f l u e n t f r a c t i o n s were monitored a t 280 nm f o r d e t e c t i o n o f g l o b i n c h a i n s . 51 F. A m i n o e t h y l a t i o n o f Separated G l o b i n Chains A m i n o e t h y l a t i o n o f the s u l f h y d r y l group o f c y s t e i n y l r e s i d u e of pep t i d e s w i t h B-bromoethylamine was f i r s t i n t r o -duced by L i n d l e y (117). The 8 - a m i n o e t h y l c y s t e i n y 1 d e r i v a t i v e from m o d i f i c a t i o n r e a c t i o n has a s t r u c t u r e s i m i l a r t o l y s i n e , and t r y p s i n can cle a v e the pe p t i d e bond formed by the c a r b o x y l group o f S- a m i n o e t h y l c y s t e i n e . More r e c e n t l y , e t h y l e n i m i n e , the r e a c t i v e i n t e r m e d i a t e formed from B-bromoethylamine, has been used by R a f t e r y and Cole (118) i n order t o o b t a i n q u a n t i t a t i v e S - aminoethylation. R R 1 a l k a l i I I -NH-CH-CO-NH-CH-CO-NH-CH-CO... + H 2 C — C H 2 • \ / CH 2 N 8M urea I i SH H room temperature c y s t e i n y l p e p t i d e e t h y l e n i m i n e R R 1 . R I I t r y p s i n r -NH-CH-CO-NH-CH-CO-NH-CH-CO... > -NH-CH-CO-NH-CH-COOH I I CH 2 CH 2 I I S S » I CH 2 CH 2 I I CH 2 CH 2 NH 3 NHT B - a m i n o e t h y l c y s t e i n y l p e p t i d e + R 1 I NH2-CH-CO... I t i s w e l l known t h a t t r y p t i c d i g e s t i o n of whole g l o b i n o r the sepa r a t e d g l o b i n chains y i e l d s about 30% o f i n s o l u b l e p e p t i d e c a l l e d "core" which co n t a i n s the c y s t e i n y l p e p t i d e s i n both a and 8-chains ( 1 4 ) . A m i n o e t h y l a t i o n o f the s u l f h y d r y l groups o f c y s t e i n y l 104 o f the a-chain o r those a t 9 3 and 112 52 o f the 3-chain, p r o v i d e s a d d i t i o n a l p o i n t s o f cleavage f o r r t-^psin reduces s u b s t a n t i a l l y the amount of t r y p t i c core (74,119). The procedure o f C l e g g , Naughton, and W e a t h e r a l l (74) f o r a m i n o e t h y l a t i o n o f separated g l o b i n c h a i n was employed. A f t e r d i s s o l v i n g the s o l i d t r i s i n the pooled f r a c t i o n of a peak from the C M - c e l l u l o s e column to a f i n a l c o n c e n t r a t i o n o f IM and a d j u s t i n g the pH to 9.2 w i t h HC1, e t h y l e n i m i n e (Matheson, Coleman & B e l l ) was added to a f i n a l c o n c e n t r a t i o n of 0.5M. Amino-e t h y l a t i o n was allowed t o proceed a t room temperature f o r 3 hours. The completion o f the r e a c t i o n was determined by the decrease to zero of the n i t r o p r u s s i d e t e s t f o r f r e e s u l f h y d r y l . The pH of the s o l u t i o n was lowered t o 3 w i t h HC1 and then the amino-ethylated, g l o b i n c h a i n was d e s a l t e d by p a s s i n g the s o l u t i o n through a column of Sephadex G-25 (coarse beads) p r e v i o u s l y e q u i l i b r a t e d w i t h 0.2M a c e t i c a c i d . The e f f l u e n t f r a c t i o n s were monitored at 280 nm and f i n a l l y the f r a c t i o n s c o n t a i n i n g the aminoethylated c h a i n (AE chain) were pooled and l y o p h i l i z e d . G. T r y p t i c D i g e s t i o n E i t h e r aminoethylated (AE) or n o n - s u b s t i t u t e d g l o b i n c h a i n was d i s s o l v e d to 20 mg/ml i n water and 2% N I U H C O 3 added i n equal volume. This l e d to some p r e c i p i t a t i o n . D i g e s t i o n was a t 37° C f o r 2 1/2 hours (3-chain) or 3 hours (a-chain) w i t h TPCK-trypsin (Cal Biochem) a t an enzyme s u b s t r a t e r a t i o of 1/100. A f t e r d i g e s t i o n , the s o l u t i o n was l y o p h i l i z e d . 53 H. P e p t i d e Mapping A comparison of p e p t i d e maps between normal and v a r i a n t c h a i n d i g e s t s was made, two dimensional h i g h v o l t a g e e l e c t r o -p h o r e s i s (HVE) and descending chromatography. Two m i l l i g r a m s o f p e p t i d e s were a p p l i e d a t the o r i g i n , 16 cm from the anode margin o f Whatman No. 3 paper and sepa r a t e d by h i g h v o l t a g e e l e c t r o p h o r e s i s at pH 6.5 i n p y r i d i n e - a c e t i c - w a t e r system (25:1:225 by volume) u n t i l the b a s i c methyl green marker reached 33 cm from the o r i g i n . The u n s t a i n e d s t r i p from the HVE sheet was then sewn i n t o a f r e s h sheet and separated a t r i g h t angles by descending chromatography w i t h n-butanol-a c e t i c a c i d - w a t e r - p y r i d i n e b u f f e r (15:3:12:10 by volume) (120) f o r 18 hours. A drop o f i n k from a P e n t e l pen served as a good v i s i b l e marker f o r chromatography. I . P e p t i d e S t a i n i n g (121,122,123) N i n h y d r i n , cadraiun n i n h y d r i n and s p e c i a l s t a i n s f o r methionine, t y r o s i n e , h i s t i d i n e , a r g i n i n e , and tryptophan were used f o r v i s u a l i z i n g p a r t i c u l a r p e p t i d e as f o l l o w s : 1. S t a i n i n g f o r methionine ( c h l o r o p l a t i n a t e s t a i n ) S o l u t i o n A - 0.002M c h l o r o p l a t i n i c a c i d ( H 2 P t C l G ' 6 H 2 0 ) (1 mg/ml H 20) S o l u t i o n B - 0.1N K l ( f r e s h l y prepared) S o l u t i o n C - 2N HCl The reagents were mixed j u s t b e f o r e use i n the f o l l o w i n g r a t i o and sequence, A:B:C = 4:0.25:0.4 ml and 76 ml of acetone 54 was added. The paper was dipped and d r i e d . A p o s i t i v e t e s t f o r t h i o e s t e r s u l f u r was a bleached area on a pink background. 2. Pauly s t a i n f o r h i s t i d i n e and t y r o s i n e S o l u t i o n A - 1% s u l f a n i t i c a c i d i n 1.2N HCl S o l u t i o n B - 5% NaN0 2 S o l u t i o n C - 10% N a 2 c 6 3 The s o l u t i o n s were kept i n a r e f r i g e r a t o r . One p a r t o f s o l u t i o n s A and B were mixed and c h i l l e d i n an i c e bath f o r 3-5 minutes, then two p a r t s o f s o l u t i o n C was added. The paper was sprayed w i t h uniform w e t t i n g on both s i d e s . The background s t a i n c o u l d be minimized by washing the chromatogram w i t h methanol. H i s t i d i n e p e p t i d e s gave a c h e r r y - r e d c o l o r w h i l e t y r o s i n e p e p t i d e s gave a brownish p u r p l e c o l o r . 3. T y r o s i n e s t a i n S o l u t i o n A - 0.1% a n i t r o s o - 6 n a p h t h o l i n acetone S o l u t i o n B - 10% HN0 3 i n acetone ( f r e s h l y prepared) The paper was dipped i n s o l u t i o n A and d r i e d . Then i t was a g a i n immersed i n s o l u t i o n ,B. A f t e r the paper s t o o d i n the fume hood f o r 5 minutes i t was heated a t 70° C f o r 3-5 minutes. A p o s i t i v e r e a c t i o n f o r t y r o s i n e showed a pink spot on the l i g h t y e l l o w background. 4. Tryptophan s t a i n S o l u t i o n A - 2% p-dimethyl aminobenzaldehyde i n acetone S o l u t i o n B - 10% HCl i n acetone The s o l u t i o n A and B were f r e s h l y prepared. The paper was dipped i n s o l u t i o n A and d r i e d , then dipped i n s o l u t i o n B. Tryptophan p e p t i d e gave a p u r p l e c o l o r which faded r a p i d l y . 55 5. Sakaguchi s t a i n f o r a r g i n i n e S o l u t i o n A - 0.2% 8-hydroxy q u i n o l i n e i n acetone S o l u t i o n B - 0.3% bromine (V/V) i n 0.5 N NaOH ( f r e s h l y prepared) A f t e r immersing the paper i n s o l u t i o n A, i t was d r i e d thoroughly w i t h a hot a i r d r y e r , then dipped i n s o l u t i o n B. A p o s i t i v e r e a c t i o n f o r a r g i n i n e was a t r a n s i e n t orange s p o t . 6. N i n h y d r i n and cadmium n i n h y d r i n s t a i n G e n e r a l l y , 0.2% n i n h y d r i n i n acetone was used t o s t a i n the chromatogram f o r p e p t i d e v i s u a l i z a t i o n . The r e a c t i o n c o u l d be a c c e l e r a t e d by h e a t i n g i n the oven at 60° f o r 20 minutes. Most p e p t i d e s g i v e p u r p l e spots but some show s p e c i a l c h a r a c t -e r i s t i c s , f o r example, when g l y c i n e i s N t e r m i n a l as i n aTpVII, there i s f i r s t a y e l l o w c o l o r which turns p u r p l e a f t e r a few hours. P e p t i d e s w i t h v a l i n e as N t e r m i n a l e.g. aTpIX, STpIII g i v e very poor and slow r e a c t i o n compared t o the o t h e r p e p t i d e s . However, the most s e n s i t i v e d e t e c t i o n o f p e p t i d e s p r e s e n t i n only s m a l l amounts i s by the cadmium n i n h y d r i n s t a i n . T h i s s o l u t i o n i s prepared by mixing 85 ml o f 0.5% n i n h y d r i n i n acetone and 15 ml of cadmium a c e t a t e s o l u t i o n (5 gm cadmium a c e t a t e + 250 ml a c e t i c a c i d + 500 ml H 2 0 ) . The paper i s dipped i n t h i s s o l u t i o n and then heated and p e p t i d e s develop as r e d d i s h - p i n k s p o t s . On o c c a s i o n , i t was u s e f u l t o employ s e v e r a l s p e c i f i c s t a i n s i n sequence. A good combination i s the c h l o r o p l a t i n a t e s t a i n f o r methionine f i r s t , f o l l o w e d by 0.2% n i n h y d r i n f o r 56 g e n e r a l p e p t i d e v i s u a l i z a t i o n and f i n a l l y the Pauly s t a i n f o r l o c a t i o n of h i s t i d i n e and t y r o s i n e p e p t i d e s . The s t a i n s f o r a r g i n i n e , tryptophan o r t y r o s i n e a l s o worked w e l l a f t e r 0.2% n i n h y d r i n . J . I s o l a t i o n Of P e p t i d e s On A P r e p a r a t i v e S c a l e Peptides c o u l d be i s o l a t e d p r e p a r a t i v e l y by high v o l t a g e e l e c t r o p h o r e s i s . The p e p t i d e a f t e r s o l u t i o n i n a b u f f e r at approximately 40 mg/ml was a p p l i e d a t 2 mg/cm on a sheet of Whatman No. 3 mm paper. B u f f e r systems employed were p y r i d i n e - a c e t i c acid-water, pH 6.5 (25:1:225 by volume); p y r i d i n e - a c e t i c acid-water, pH 3.6 (1:10:89 by volume) and formic a c i d - a c e t i c acid-water, pH 1.9 (25:87:888 by volume). Vari o u s dyes were used as markers to f o l l o w the progress of extended p r e p a r a t i v e h i g h v o l t a g e e l e c t r o p h o r e s i s of the p e p t i d e e.g. f o r 2-3 hours a t 3 k.v. The p e p t i d e was l o c a t e d by s t a i n i n g guide s t r i p s with s p e c i a l s t a i n s , n i n h y d r i n or cadmium n i n h y d r i n s t a i n . The c o r r e s p o n d i n g u n s t a i n e d paper s t r i p was sewn i n t o a f r e s h sheet and r e - r u n by HVE i n another b u f f e r system. A f t e r l o c a t i n g the p e p t i d e by guide s t r i p s t a i n i n g , the u n s t a i n e d p e p t i d e on the remainder of the paper was e l u t e d u s i n g 0.01 N NHi»OH and the e l u a n t l y o p h i l i z e d . K. Cyanogen Bromide Cleavage Of Methionine C o n t a i n i n g Peptides The chemical cleavage o f methionyl peptides with cyanogen bromide was f i r s t d e s c r i b e d by Gross and Witkop (124) and i t 57 cleavage o f p r o t e i n s and p e p t i d e s . The chemical r e a c t i o n i s b e l i e v e d t o be as f o l l o w s : R R 1 A c i d R R 1 • 1 1 1 -NH-CH-CO-NH-CH-C-NH-CH-CO- + Br-C=N —> NH-CH-CO-NH-CH-C-NH-CH-CO-y II • , » j / CH 2 0 CH 2 0 CH2-S-CH3 Cyanogen bromide CH 2-S-CH 3 /© ' Br C=N R R © R 1 -NH-CH-CO-NH-CH-C=0 <c . -NH-CH-CO-NH-CH-C=NH-CH-CO / » / \ CH 2 0 CH 2 0 R<9 CH 2 CH 2 Homoserine Lactone + CH3-SCN e R 1 + N H 3-CH-CO-Under a c i d i c c o n d i t i o n s and w i t h excess o f cyanogen bromide, the cleavage i s very s p e c i f i c f o r methionine, although unsub-s t i t u t e d c y s t e i n y l r e s i d u e s can be o x i d i z e d t o c y s t e i c a c i d but t h i s s i d e r e a c t i o n i s a minor one compared t o the methionyl cleavage (125) and can be prevented by p r i o r m o d i f i c a t i o n o f the s u l f h y d r y l group. Under the c o n d i t i o n s f o r complete a c i d h y d r o l y s i s o f peptides^homoserine l a c t o n e i s converted i n t o an e q u i l i b r i u m mixture w i t h homoserine. Fo r convenience, cyanogen bromide was d i s s o l v e d i n 70% for m i c a c i d t o a c o n c e n t r a t i o n o f 40 mg/ml. A c a l c u l a t e d amount was added t o d i s s o l v e the p e p t i d e and 70% f o r m i c a c i d was then added t o make the f i n a l s o l u t i o n , 10 mg/ml. The s o l u t i o n was c a r e f u l l y stoppered and inc u b a t e d a t room temperature f o r 24 hours. The excess reagent was removed by l y o p h i l i z a t i o n . 58 L. D i l u t e A c e t i c Cleavage Of P e p t i d e s B u l l , Hahn and B a p t i s t (126) d i s c o v e r e d t h a t the a s p a r t y l l i n k a g e i n p e p t i d e chains i s very s e n s i t i v e t o a c i d h y d r o l y s i s on the b a s i s of an e a r l y r e l e a s e o f f r e e a s p a r t i c a c i d a f t e r t r e a t m e n t A t h e p r o t e i n w i t h m i l d a c i d . Subsequently, P a r t i d g e and Davis (127) d e s c r i b e d a method f o r r e f l u x i n g a p r o t e i n w i t h 0.25 M a c e t i c a c i d at 100° C which r e s u l t e d i n an almost completely s e l e c t i v e r e l e a s e o f f r e e a s p a r t i c a c i d from p r o t e i n s . Furthermore, s t u d i e s o f 0.03 N HCl h y d r o l y s i s of p r o t e i n s of known sequence confirmed t h i s s p e c i f i c i t y . However, the a s p a r a g i n y l p e p t i d e bonds were a l s o h y d r o l y z e d b u t a t a slower r a t e than the a s p a r t y l l i n k a g e s (128). I t i s now c l e a r t h a t d i l u t e a c e t i c a c i d h y d r o l y s i s of p r o t e i n leads to a s p e c i f i c cleavage o f a s p a r t y l r e s i d u e s as w e l l as a slow c o n v e r s i o n of a s p a r a g i n y l r e s i d u e s t o a s p a r t y l which are then c l e a v e d (129) . In the p r e s e n t work, the method a c c o r d i n g t o P a r t i d g e and Davis (127) was employed and the a s p a r t i c a c i d - c o n t a i n i n g p e p t i d e was d i s s o l v e d i n 0.25 M a c e t i c a c i d ( f r e s h l y prepared) and h y d r o l y z e d at 110° C f o r 24 hours under vacuum. The h y d r o l y -s a t e was then d r i e d i n a r o t a r y Evapo-Mix (Buchler Instruments). M. N - t e r m i n a l Amino A c i d A n a l y s i s By D a n s y l a t i o n Method FDNB ( l - f l u o r o - 2 , 4-dinitrobenzene) was i n t r o d u c e d by Sanger (130) as the f i r s t micro-method f o r determining the N - t e rminal r e s i d u e i n p r o t e i n s and p e p t i d e s . R ecently, Gray 59 and H a r t l e y (131) d e s c r i b e d a new f l u o r e s c e n t reagent *- Dansyl c h l o r i d e o r DNS-C1 (1-dimethylaminonaphthalene-5-sulfonyl c h l o r i d e ) which i n c r e a s e s the s e n s i t i v i t y of the r e a c t i o n 100 f o l d . The chemical r e a c t i o n i s the f o l l o w i n g : H 3C SOz-Cl DNS-C1 R R + H2N-CH-CO-NH-CH-pe p t i d e H,C \ /CH 3 S02-NH-CH-CO-NH-CH— A c i d h y d r o l y s i s H H3C. R I S02-NH-CH-COOH DNS - amino a c i d The DNS-amino a c i d s can be sepa r a t e d e i t h e r by paper e l e c t r o p h o r e s i s or t h i n l a y e r chromatography and l o c a t e d and i d e n t i f i e d by t h e i r very i n t e n s e f l u o r e s c e n c e under U.V. l i g h t . D a n s y l a t i o n o f peptides was performed as d e s c r i b e d by Gray (132). About 0.005-0.01 umole o f the p e p t i d e , a f t e r d r y i n g , was. d i s s o l v e d i n 20 m i c r o l i t r e s o f 0.1M NaHC0 3 and again evaporated t o e l i m i n a t e ammonia. The r e s i d u e was then d i s s o l v e d i n 10 m i c r o l i t r e s o f d i s t i l l e d water and 10 m i c r o l i t r e s o f DNS-Cl (2.5 mg/ml i n acetone) added. The tube was s e a l e d w i t h p a r a f i l m and incubated at 37° C f o r 2 hours. The DNS-peptide s o l u t i o n was d r i e d i n a r o t a r y Evapo-Mix and h y d r o l y z e d with constant b o i l i n g 5.7N HCl under vacuum over n i g h t . A f t e r removal o f the a c i d under vacuum, the h y d r o l y s a t e 60 was d i s s o l v e d i n 2M N H 4 O H b e f o r e a p p l i c a t i o n to S i l i c a G e l G TLC p l a t e to i d e n t i f y the DNS-amino a c i d . DNS-amino a c i d s were separated by t h i n l a y e r chromatography a c c o r d i n g to Black and Dixon (133). For s e p a r a t i o n o f a p o l a r DNS-amino a c i d s the c h l o r o f o r m - m e t h a n o l - a c e t i c a c i d s o l v e n t (95:10:1 by volume) was used w h i l e f o r p o l a r DNS-amino a c i d s ; N-propanol-ammonium hydroxide s o l v e n t (90:20 by volume) was employed. Chromatography was f o r 2 and 4 hours r e s p e c t i v e l y . The DNS-amino a c i d s were l o c a t e d under U.V. l i g h t and i d e n t i f i e d by comparison with s t a n d a r d DNS-amino a c i d s . The TLC p l a t e s were photographed under U.V. l i g h t with p o l a r o i d f i l m (30 00 ASA) u s i n g a U.V. f i l t e r . N. Leucine Amino P e p t i d a s e (LAP) H y d r o l y s i s Of A P e p t i d e LAP, an exopeptidase, r e l e a s e s amino a c i d s s e q u e n t i a l l y from the N-terminus o f a p o l y p e p t i d e c h a i n when the a-amino group i s f r e e . N - terminal p r o l i n e i s only very s l o w l y h y d r o l y z e d but a t h i g h c o n c e n t r a t i o n o f enzyme and a f t e r long p e r i o d s of d i g e s t i o n , some p r o l i n e and the subsequent amino a c i d s are r e l e a s e d ' i n t r a c e s . At low enzyme s u b s t r a t e r a t i o s w i t h a s h o r t i n c u b a t i o n and i n the absence of p r o l i n e , the k i n e t i c s of r e l e a s e o f amino a c i d s can p r o v i d e i n f o r m a t i o n on the sequence of the N - terminal r e g i o n . In a d d i t i o n , the amide amino a c i d s , asparagine and glutamine, are r e l e a s e d i n t a c t and i t can o f t e n be determined whether these r e s i d u e s are p r e s e n t . For LAP d i g e s t i o n , a pept i d e was d i s s o l v e d i n 0.2M ammonium ace t a t e b u f f e r c o n t a i n i n g 0.005M MgCl2, pH 8.5 (134) and the LAP was then added a t a enzyme s u b s t r a t e r a t i o of 1:100. The s o l u t i o n was in c u b a t e d a t 37° C f o r 18 hours. The d i g e s t was d r i e d and an a l i q u o t a p p l i e d d i r e c t l y t o the amino a c i d a n a l y z e r . 0. C - t e r m i n a l Amino A c i d D etermination By Carboxypeptidase A Carboxypeptidase A, one of the exopeptidase from pancreas, i s w e l l known to r e l e a s e amino a c i d s s e q u e n t i a l l y from the C-terminus of a p e p t i d e . Amino a c i d s w i t h aromatic or a l i p h a t i c s i d e chains are r e l e a s e d r a p i d l y but g l y c i n e and the a c i d i c amino a c i d s are r e l e a s e d s l o w l y . No h y d r o l y s i s i s d e t e c t a b l e when a r g i n i n e o r p r o l i n e i s the C - t e r m i n a l amino a c i d . However, i n a p p r o p r i a t e circumstances, i t i s p o s s i b l e t o deduce the C - t e r m i n a l sequence by a k i n e t i c study o f amino a c i d s r e l e a s e d from a p e p t i d e . The k i n e t i c s o f r e l e a s e o f amino a c i d s from peptides by carboxypeptidase A was performed a c c o r d i n g t o Amber (135). F i v e m i c r o l i t r e s of carboxypeptidase A-DFP, 4 4 mg/ml (Worthington Biochem. Corp) was c e n t r i f u g e d t o sediment the carboxypeptidase c r y s t a l s which were resuspended and washed t h r e e times with c o l d water to remove endogenous f r e e amino a c i d s . The washed c r y s t a l s were then d i s s o l v e d i n 50 m i c r o l i t r e s o f 2M N H 4 H C O 3 . Twenty m i c r o l i t r e s o f enzyme s o l u t i o n was added t o the pept i d e s o l u t i o n (0.09 umoles i n 0.289 ml of water) t o a f i n a l c o n c e n t r a t i o n 62 0.2M NHifHCO 3, pH 8.6 i n 0.3 ml o f d i g e s t . The s o l u t i o n was incubated at 37° C and 0.1 ml a l i q u o t s were taken a t 30,90, and 270 minutes. Enzyme a c t i v i t y was terminated by a d d i t i o n of g l a c i a l a c e t i c a c i d and the samples were d r i e d under vacuum. In or d e r to remove ammonium a c e t a t e completely, the dry r e s i d u e was r e d i s s o l v e d i n water and d r i e d a g a i n , t h i s b e i n g repeated 3-4 times b e f o r e d i s s o l v i n g the sample i n pH 2.2 b u f f e r f o r a p p l i c a t i o n t o the amino a c i d a n a l y z e r . P. Amino A c i d A n a l y s i s Peptides which were e l u t e d d i r e c t l y w i t h 6N HCl from a peptid e map s t a i n e d w i t h 0.02% n i n h y d r i n , were h y d r o l y z e d , a f t e r e v a c u a t i o n a t the o i l pump and s e a l i n g a t 110° C f o r 24 hours. More c r i t i c a l amino a c i d analyses of pep t i d e s were done a f t e r l y o p h i l i z a t i o n o f d i l u t e NH(,OH e l u a t e s of the pe p t i d e from u n s t a i n e d paper a f t e r guide s t r i p l o c a t i o n . These e l u a t e s were h y d r o l y z e d i n 0.5 - 1 ml of cons t a n t b o i l i n g 5.7N HCl a t 110° C f o r 24 hours a f t e r e v a c u a t i o n a t o i l pump and s e a l i n g . The h y d r o l y s a t e s were d r i e d under vacuum, d i s s o l v e d i n pH 2.2 b u f f e r and a p p l i e d t o the Beckman amino a c i d a n a l y z e r Model 120C u s i n g a s i n g l e column system as d e s c r i b e d by Devenyi (136), Hew and Dixon (137). The r e s o l u t i o n o f a standar d amino a c i d run i s shown on the F i g u r e 7. Standard Amino Acid Run Single Column System Buffer A PH 3-43 0-2 N Sodium Citrate Buffer B pH 4-25 0-8N SodiumCltrote Buffer Change 70 minutes IJ \4 Figure 7. A resolution of standard amino acid run using a single column system (136,137) 64 IV. IDENTIFICATION OF TRYPTIC PEPTIDES ON PEPTIDE MAPS Two dimensional h i g h v o l t a g e paper e l e c t r o p h o r e s i s and p a r t i t i o n chromatography was f i r s t used f o r s t u d y i n g abnormal hemoglobins by Ingram (138) and was c a l l e d by him, the " f i n g e r p r i n t t e c h n i q u e " . When the t r y p t i c d i g e s t o f the whole g l o b i n from Hb A and Hb S were compared by t h i s e l e g a n t technique, only a s i n g l e p e p t i d e a l t e r a t i o n was seen. Furthermore, when the p e p t i d e was i s o l a t e d and i t s amino a c i d a n a l y s i s and sequence determined, t h i s c l a s s i c a l work showed t h a t the gl u t a m i c a c i d r e s i d u e a t p o s i t i o n 6 o f the 3-chain was r e p l a c e d i n Hb S by v a l i n e (139). T h i s work s u b s t a n t i a t e d i n the most c o n v i n c i n g way the concept o f "molecular d i s e a s e " as proposed by P a u l i n g e t a l (5). I t i s now g e n e r a l l y accepted t h a t p e p t i d e mapping o f the abnormal hemoglobin i s the most s a t i s f a c t o r y , uncomplicated and r a p i d method t o l o c a t e the s i t e o f mutation i n a p a r t i c u l a r t r y p t i c p e p t i d e o r p e p t i d e s but i t i s necessary t h a t the p e p t i d e maps prepared from normal hemoglobin are r e p r o d u c i b l e and the p o s i t i o n s o f the known p e p t i d e s A r e c o g n i z e d and accounted f o r . In the o r i g i n a l p e p t i d e maps u s i n g HVE a t pH 6.4 and ascending chromatography i n n - b u t a n o l - a c e t i c acid-water system t o study the whole g l o b i n , the t r y p t i c p e p t i d e s were not w e l l r e s o l v e d , but B a g l i o n i (140) d e s c r i b e d a s o l v e n t c o n s i s t i n g of iso-amyl a l c o h o l - p y r i d i n e - w a t e r system f o r the second dimension which improved the r e s o l u t i o n o f the pe p t i d e s and the sharpness of the spots c o n s i d e r a b l y . Analyses of p e p t i d e s on chromatogram 65 u s i n g t h i s system was subsequently e s t a b l i s h e d (140) and accepted as a r e f e r e n c e f o r the whole g l o b i n p e p t i d e map. However, the chromatogram o f unseparated g l o b i n chains appears r a t h e r complicated due t o the presence o f over 30 t r y p t i c p e p t i d e s from both a and 8-chains. Recently C l e g g , e t a l (74) p u b l i s h e d a method o f c h a i n s e p a r a t i o n and d e s c r i b e d the t r y p t i c p e p t i d e analyses of s e p a r a t e d a and 3-chains u s i n g HVE pH 4.7 and descending chromatography i n n - b u t a n o l - a c e t i c a c i d - w a t e r -p y r i d i n e system (120). However, f o r the p r e s e n t s t u d i e s , HVE pH 6.5 f o l l o w e d by descending chromatography was found t o g i v e the b e s t p e p t i d e map. As t h i s map uses a d i f f e r e n t system from t h a t o f Clegg e t a l (74) analyses o f p e p t i d e s appearing on the chromatogram were r e q u i r e d i n o r d e r t o e s t a b l i s h a normal p a t t e r n w i t h which to compare the p a t t e r n s f o r abnormal hemoglobins. A A. Analyses Of P e p t i d e s On The Chromatogram Of Non AEg The a c h a i n o b t a i n e d from the f r a c t i o n a t i o n of normal g l o b i n by C M - c e l l u l o s e chromatography (74) was d i r e c t l y d e s a l t e d on Sephadex G-25 (coarse beads) w i t h 0.2M a c e t i c a c i d and the A p r o t e i n peak l y o p h i l i z e d . The non AEa c h a i n was d i g e s t e d w i t h T P C K - t r y p s i n a t enzyme s u b s t r a t e r a t i o 1 t o 100 f o r 3 hours and the r e a c t i o n was terminated by l y o p h i l i z a t i o n . A f t e r d i s s o l v i n g 2 mg of the t r y p t i c p e p t i d e i n h i g h v o l t a g e b u f f e r , the supernatant o b t a i n e d on c e n t r i f u g a t i o n was p e p t i d e mapped u s i n g HVE pH 6.5 and descending chromatography i n n - b u t a n o l - a c e t i c a c i d - w a t e r - p y r i d i n e system (120). 66 F o r s p e c i a l s t a i n i n g , t h r e e chromatograms were prepared. A combination o f c h l o r o p l a t i n a t e s t a i n f o r methionine f o l l o w e d by 0.2% n i n h y d r i n s t a i n f o r g e n e r a l p e p t i d e s and f i n a l l y Pauly s t a i n f o r t y r o s i n e and h i s t i d i n e c o n t a i n i n g p e p t i d e s were employed on the f i r s t chromatogram. The o t h e r two p e p t i d e maps were both s t a i n e d w i t h 0.2% n i n h y d r i n i n i t i a l l y ; the second was f o l l o w e d by Sakaguchi s t a i n f o r a r g i n y l p e p t i d e w h i l e the t h i r d chromatogram was t r e a t e d w i t h E h r l i c h reagent f o r tryptophan c o n t a i n i n g p e p t i d e . A f t e r n i n h y d r i n s t a i n i n g , 12 major p e p t i d e s were ob t a i n e d d e s i g n a t e d as A,B,C,D,E,F,G,H,I,J,K,L and a t l e a s t 4 minor p e p t i d e s ; M,N,0,P were a l s o d e t e c t a b l e ( P l a t e 8 and F i g . 9 ) . The r e s u l t s o f s p e c i a l s t a i n i n g are a l s o shown on F i g . 9. S i n c e only two t r y p t i c p e p t i d e s , aTpV and ctTpIX of a-chain (see F i g . 10) are methionine c o n t a i n i n g p e p t i d e s , h i s t i d y l r e s i d u e s b e i n g p r e s e n t i n the l a t t e r , aTpV would be expected to show p o s i t i v e s t a i n i n g f o r methionine w h i l e the aTpIX would be p o s i t i v e f o r both c h l o r o p l a t i n a t e and Pauly s t a i n s . From the F i g . 9, E and B p e p t i d e s appear to correspond t o aTpV and IX r e s p e c t i v e l y . The minor M p e p t i d e , l i k e the B p e p t i d e was p o s i t i v e f o r the methionine-Pauly s t a i n , however i t s m o b i l i t y appeared more b a s i c . T h i s p e p t i d e i s thought to be aTpVIII-IX, a r i s i n g from a p a r t i a l cleavage by t r y p s i n . A c c o r d i n g to the s p e c i a l s t a i n i n g ( F i g . 9 ) , the G p e p t i d e was o n l y p o s i t i v e f o r Sakaguchi s t a i n w h i l e A - a c i d i c and H-basic p e p t i d e , both gave p o s i t i v e c o l o r on Sakaguchi and Pauly s t a i n s . The 6 7 HVE pH 6.5 P l a t e 8. A t r y p t i c p e p t i d e map of non AEa w i t h 0.2% n i n h y d r i n s t a i n . 68 3 Cl* .Oo O P F i g u r e 9 0 ^ ^ 6 81-90 © nj) 0 137-139 F i g u r e 9a Origin Anode F i g u r e 9. A diagram o f p e p t i d e maps o f P l a t e 8. The p e p t i d e s were d e s i g n a t e d by l e t t e r s and a l s o numbered a c c o r d i n g t o t h e i r p o s i t i v e r e a c t i o n t o s p e c i a l s t a i n s ; 1 = c h l o r o p l a t i n a t e , 2 = P a u l y , 3 = t r y p t o p h a n and 4 = S a k a g u c h i s t a i n . The b r o k e n l i n e i n d i c a t e s a minor p e p t i d e . F i g u r e 9a. A diagram o f the t r y p t i c p e p t i d e map c o r r e s p o n d i n g t o t r y p t i c p e p t i d e number on t h e b a s i s o f amino a c i d a n a l y s e s and s p e c i a l s t a i n s . aTpI otTpII .aTpIII (3) 7al-Leu-Ser-Pro-Ala-Asp-Lys Thr-Asn-Val-Lys A l a - A l a - T r y - G l y - L y s 1 7 8 11 12 16 otTpIV (2,4) aTpV (1) f a l - G l y - A l a - H i s - A l a - G l y - G l u - T y r - G l y - A l a - G l u - A l a - L e u - G l u - A r g Met-Phe-Leu-Ser-Phe-Pro-Thr-Thr-Lys L7 31 32 40 aTpVI (2) gTpVII (2) gTpVIII rhr-Tyr-Phe-Pro-His-Phe- Asp-Leu-Ser-His-Gly-Ser-Ala-Gin-Val-Lys G l y - H i s - G l y - L y s Lys U 56 57 60 61 aTpIX (1,2) 7al-Ala- Asp- A l a - Leu-Thr-Asn-Ala-Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn- A l a - Leu-Ser-Ala-Leu-Ser-Asp-Leu-His -Ala-His-Ly£ >2 68 74 76 9( aTpX (4) gTpXI Leu-Arg Val-Asp-Pro-Val-Asn-Phe-Lys 91 92 93 99 gTpXII (core)(2) jeu-Leu-Ser-His-Cys-Leu-Leu-Val-Thr-Leu-Ala-Ala-His-Leu-Pro-Ala-Glu-Phe-Thr-Pro-Ala-Val-His-Al a-Ser-Leu-Asp-Lys LOO 104 105 12' gTpXIII (core) gTpXIV (2,4) ?he-Leu-Ala-Ser-Val-Ser-Thr-Val-Leu-Thr-Ser-Lys Tyr-Arg L28 139 140 141 ri g u r e 10. Amino a c i d sequences of t r y p t i c peptide of Non AE a-chain (37) . The number i n p a r e n t h e s i s a f t e r the t r y p t i c p e p t i d e number i n d i c a t e s p o s i t i v e f o r s p e c i a l s t a i n i n g . 1 = c h l o r o p l a t i n a t e , 2 = Pauly s t a i n , 3 = tryptophan s t a i n , 4 = Sakaguchi s t a i n . o 70 a r g i n i n e c o n t a i n i n g p e p t i d e s of t r y p t i c a c h a i n are known t o be aTpX, IV and XIV ( F i g . 10) and i t i s a l s o known t h a t aTpIV-a c i d i c and X l V - b a s i c p e p t i d e both c o n t a i n t y r o s i n e . T h e r e f o r e , the G, A and H p e p t i d e r e p r e s e n t aTpX, IV and XIV r e s p e c t i v e l y . The I p e p t i d e showed p o s i t i v e s t a i n i n g f o r tryptophan c o r r e s -ponding to aTpIII which i s the o n l y p e p t i d e c o n t a i n i n g tryptophan i n the a-chain ( F i g . 10). F i n a l l y amino a c i d analyses were performed on the chromatographed p e p t i d e s o b t a i n e d A from 5 mg t r y p t i c p e p t i d e o f the non AEa c h a i n . A f t e r s t a i n i n g w i t h 0.02% n i n h y d r i n and h e a t i n g to 60° C f o r 20 minutes, the p e p t i d e spots were cut and d i r e c t l y e l u t e d w i t h 6N HCl. The e l u a t e was h y d r o l y z e d at 110° C f o r 24 hours under vacuum. A h y d r o l y s a t e o f a major p e p t i d e was q u a n t i t a t i v e l y enough f o r one a n a l y s i s but i n the case o f a minor p e p t i d e , the e l u a t e s from two paper chromatograms were r e q u i r e d . The h y d r o l y s a t e was a p p l i e d t o a Beckman amino a c i d a n a l y z e r Model 120C u s i n g a s i n g l e column system (136,137). The r e s u l t s of amino a c i d analyses of the p e p t i d e s are shown on T a b l e I I . The p e p t i d e s : A,B,E,G, and H being b e l i e v e d t o r e p r e s e n t aTpIV, IX, V, X, and XIV r e s p e c t i v e l y on the b a s i s o f s p e c i a l s t a i n i n g were a l s o confirmed by the amino a c i d a n a l y s e s . The I-tryptophan p o s i t i v e p e p t i d e showed the amino a c i d composition s i m i l a r to aTpIII except the absence o f tryptophan which was d e s t r o y e d d u r i n g a c i d h y d r o l y s i s . In summary i t would appear from amino a c i d analyses and s p e c i a l s t a i n i n g t h a t the 12 major p e p t i d e s : D,J,I,A,E,F,L,K,B,G,C and H Table I I . Amino a c i d analyses o f t r y p t i c peptides from the peptide map of non AEpt A B C D E umoles res i d u e s umoles r e s i d u e s umoles res i d u e s umoles residues umoles r e s i d u e s Lys 6.019 1.00 (1) 0 .116 0 .86 (1) 0 .060 0.92 (1) 0 .027 0.91 (1) His 0.10 2 0.77 (1) 0.050 2.63 (3) Arg 0.122 0.92 (1) Asp 0.114 6 .00 (6) 0 .323 2 .30 (2) 0.069 1.06 (1) MetSG-2 t r a c e (1) t r a c e (1) Thr 0 .020 1.05 (1) 0 .045 1.85 (2) Ser 0.034 1.79 (2) 0.065 1.00 (1) 0.034 1.12 (1) Glu 0.409 3.09 (3) Pro 0.021 1.05 (1) 0 .099 0 .73 (1) 0.043 0.66 (1) 0 .019 0.65 (1) Gly 0.436 3.30 (3) A l a 0.540 4.09 (4) 0.132 6.95 (7) 0 .076 1.16 (1) V a l 0.127 0.96 (1) 0.061 3.20 (3) 0 .265 1 .96 (2) 0 .062 0.95 (1) Met • Leu 0.120 0.91 (1) 0.074 3.89 (4) 0 .058 0.89 (1) 0.036 1.20 (1) Tyr 0.049 0.40 (1) Phe 0 .107 0 .79 (1) 0.058 1.92 (2) corresponding t r y p t i c p e p t i d e IV IX XI . I v" -' R e f r a c t i o n a t e d i n HVE pH 3.6 *Homoserine l a c t o n e **Homoserine' T a b l e I I . Amino a c i d analyses o f t r y p t i c p e p t i d e s from the pept i d e map o f non AEa (continued) F G H I * J umoles r e s i d u e s umoles r e s i d u e s umoles res i d u e s umoles r e s i d u e s umoles r e s i d u e s Lys 0.110 0.89 (1) 0 .110 0.97 (1) 0.149 0.97 (1) His 0.206 1.67 (2) Arg 0 .087 1.06 (1) 0.109 1.00 (1) Asp 0.124 1.01 (1) 0.166 1.08 (1) MetS0 2 Thr 0.127 1.03 (1) 0.128 0.84 (1) Ser 0.283 2.30 (2) Glu 0.143 1.16 (1) Pro 0.083 0.67 (1) Gly 0.131 1.06 (1) 0 .115 1.02 (1) A l a 0.132 1.07 (1) 0 .225 1.99 (2) V a l 0.109 0.87 (1) 0.169 1.10 (1) Met , Leu 0.120 0.98 (1) 0.076 0 .93 (1) T y r 0.040 0.33 (1) 0 .044 0.40 (1) Phe 0.233 1.89 (2) c o r r e s p o n d i n g t r y p t i c VI X XIV I I I I I p e p t i d e  *nos i +• -i UP «ir.fii n -for trvnfnnhAh A Table I I . Amino a c i d analyses o f t r y p t i c p e p t i d e s from the p e p t i d e map of non AEa (continued) K L N O P ymoles r e s i d u e s umoles r e s i d u e s umoles r e s i d u e s ymoles r e s i d u e s umoles r e s i d u e s Lys 0.025 1.00 (1) 0.061 1.19 (1) 0.047 1.02 (1) 0.049 1.81 (2) 0.059 0.92 (1) His 0.029 0.57 (1) 0.065 1.50 (2) Arg Asp 0.047 1.02 (1) 0.043 1.60 (2) MetS0 2 Thr 0.033 1.22 (1) 0.062 0.99 (1) Ser 0.099 2.15 (2) 0.032 1.21 (1) 0.072 1.12 (1) Glu Pro 0.019 0.71 (1) Gly 0.111 2.17 (2) A l a 0.113 2.40 (2) 0.034 1.26 (1) V a l 0.053 1.96 (2) Met Lem 0.087 1.89 (2) 0.023 0.89 (1) Tyr Phe c o r r e s pondi ng t r y p t i c V I I I VII 81-90 I - I I 137-139 pe p t i d e  74 corresponded t o aTpI, I I , I I I , IV, V, VI, V I I , V I I I , IX, X, XI and XIV r e s p e c t i v e l y (Table I I , F i g . 9 and 9 a ) . aTpXII and X I I I which are known t o remain i n the i n s o l u b l e core a f t e r t r y p t i c d i g e s t , d i d not appear i n t h i s s t udy. The a n a l y s i s of M p e p t i d e was not o b t a i n e d due t o mechanical problems w i t h the a n a l y z e r d u r i n g the run. However, based on the s p e c i a l s t a i n i n g and m o b i l i t y of the M p e p t i d e i t i s thought to be TpVIII-IX as p r e v i o u s l y mentioned. A c c o r d i n g t o the amino a c i d analyses (Table II) the t h r e e minor p e p t i d e s ; 0, N, and P appeared t o r e p r e s e n t the a T p I - I I , the 81-90 r e s i d u e s -Ser-Ala-Leu-Ser-Asp-Leu-His-Ala-His-Lys and 137-139 r e s i d u e s -Thr-Ser-Lys o f a-chain r e s p e c t i v e l y ( F i g . 10). I t i s thought t h a t the presence o f minor p e p t i d e s ; N and P i s due to the presence of chymotrypsin contamination i n the TPCK-trypsin r e s u l t i n g i n the cleavage o f the C - t e r m i n a l l e u c i n e a t r e s i d u e 80 and r e s i d u e 136 of aTpIX and X I I I r e s p e c t i v e l y ( F i g . 10). I t s h o u l d a l s o be noted t h a t the amino a c i d analyses gave low y i e l d s f o r t y r o s i n e , p o s s i b l y due t o some d e g r a d a t i o n d u r i n g i s o l a t i o n of the p e p t i d e from the chromatogram. a B. Analyses Of A T r y p t i c P e p t i d e Map Of AEg Chain (aminoethylated  g A chain) The g g l o b i n s e p a r a t e d by C M - c e l l u l o s e chromatography A was aminoethylated a c c o r d i n g to Clegg e t a l (74) . The AEg was d i g e s t e d w i t h TPCK-trypsin a t an enzyme s u b s t r a t e r a t i o 1 to 100 f o r 2 1/2 hours. The s t u d i e s o f t r y p t i c p e p t i d e maps were 75 / c a r r i e d out i n the same manner as f o r the a c h a i n . The chromatogram i s shown on P l a t e 11. The major and some minor p e p t i d e s are d e s i g n a t e d by l e t t e r s ( F i g . 12). The r e s u l t s o f s p e c i a l s t a i n s are a l s o summarized on F i g . 12. Sin c e the B p e p t i d e g i v e s p o s i t i v e s t a i n f o r methionine, i t must correspond t o the BTpV p e p t i d e which i s the onl y one c o n t a i n i n g methionine i n the B-chain ( F i g . 13). The B-peptide showing the same m o b i l i t y i n e l e c t r o p h o r e s i s as the B p e p t i d e but m i g r a t i n g s l i g h t l y slower i n chromatography and a l s o n e g a t i v e to c h l o r o p l a t i n a t e s t a i n , i s b e l i e v e d to be an o x i d i z e d methionine d e r i v a t i v e o f B^pV. From s p e c i a l s t a i n i n g , the J p e p t i d e was shown p o s i t i v e f o r tryptophan w h i l e the W-a minor p e p t i d e gave a c o l o r r e a c t i o n f o r tryptophan, t y r o s i n e , and a r g i n i n e ( F i g . 12) s u g g e s t i n g i d e n t i t y w i t h the BTpIII and BTpIV p e p t i d e s r e s p e c t i v e l y ( F i g . 13). The E and M p e p t i d e s , both gave a p o s i t i v e Pauly s t a i n . In a d d i t i o n the former showed a r g i n i n e c o n t a i n i n g p e p t i d e w h i l e the l a t t e r was p o s i t i v e f o r t y r o s i n e ( F i g . 12), a p a t t e r n c o n s i s t e n t w i t h the sequences o f BTpXI and XV p e p t i d e s r e s p e c t i v e l y ( F i g . 13). The a n a l yses of p e p t i d e s from the maps were c a r r i e d out A as f o r the a c h a i n . The r e s u l t s are shown on T a b l e I I I . J - a trypotphan p o s i t i v e p e p t i d e r e v e a l e d the amino a c i d a n a l y s i s c orresponding to BTpII except f o r tryptophan which i s des t r o y e d d u r i n g the a c i d h y d r o l y s i s . As found p r e v i o u s l y , the y i e l d s o f t y r o s i n e were low, p o s s i b l y due t o o x i d a t i o n d u r i n g the i s o l a t i o n o f the p e p t i d e . A l l t y r o s i n e c o n t a i n i n g 76 HVE pH 6.5 P l a t e 11. A t r y p t i c p e p t i d e map o f AE£ w i t h n i n h y d r i n s t a i n . 77 F i g u r e 12. A diagram o f t r y p t i c p e p t i d e map from P l a t e 11. The p e p t i d e s were d e s i g n a t e d by l e t t e r s w h i l e the numbers i n d i c a t e d a p o s i t i v e r e a c t i o n t o s p e c i a l s t a i n ; 1 = c h l o r o p l a t i n a t e , 2 = P a u l y , 3 = t r y p t o p h a n , 4 = S a k a g u c h i , and 5 = t y r o s i n e s t a i n . F i g u r e 12a.The p e p t i d e diagram showing t h e c o r r e s p o n d i n g t r y p t i c p e p t i d e o f the AEg on t h e b a s i s o f amino a c i d a n a l y s e s and s p e c i a l s t a i n s . gTp! (2) gTpII (3) g T p I I I (4) /al-His-Leu-Thr-Pro-Gla-Glu-Lys S e r - A l a - V a l - T h r - A l a - L e u - T r y - G l y - L y s Val-Asn-Val-Asp-Glu-Val-Gly-Gly-Glu-Ala^[£u-Gty-Ar< 1 8 9 17 18 3( gTpIV (2,3,4,5) BTpV (1) Jeu-Leu-VaH^al-Qyr-Pro-Try-Thr-G]n-Arg Phe-Phe-Glu-Ser-Phe-Gly-Asp-Leu-Ser-Thr-Pro-Asp-Ala-Val-Met-Gly-Asn-Pro-Lys 31 40 41 5< gTpVI BTpVII (2) gTp V I I I gTpIX (2) / a l - L y s A l a - H i s - G l y - L y s Lys Va l - L e u - G l y - A l a - P h e - S e r - A s p - G l y - L e u - A l a - H i s - L e u - A s p - A s n - L e u - L y s 50 61 62 65 66 67 82 gTpX (2) gTpXI (2,4) 31y-Thr-Phe-Ala-Thr-Leu-Ser-Glu-Leu-His-SAC-Asp-Lys Leu-His-Val-Asp-Pro-Glu-Asn-Phe-Arg 33 95 96 104 'gTpXII-a gTpXII-b (2) gTpXIII (2,5) jeu-Leu-Gly-Asn-Val-Leu-Val-SAC V a l - L e u - A l a - H i s - H i s - P h e - G l y - L y s G l u - P h e - T h r - P r o - P r o - V a l - G l n - A l a - A l a - T y r - G l n - L y s L05 112 113 120 121 13: gTpXIV (2) gTpXV (2,5) / a l - V a l - A l a - G l y - V a l - A l a - A s n - A l a - L e u - A l a - H i s - L y s T y r - H i s L33 144 145 146 a r i g u r e 13. Amino a c i d sequences o f t r y p t i c p e p t i d e o f AEg . The number i n p a r e n t h e s i s a f t e r the t r y p t i c p e p t i d e number i n d i c a t e s t h e p o s i t i v e f o r s p e c i a l s t a i n (numbering as i n d i c a t e d i n F i g u r e 1 2 ) . SAC = S amino e t h y l c y s t e i n CO Table I I I . Amino a c i d analyses o f t r y p t i c peptides from the peptide map of AEg A B C D E umoles r e s i d u e s umoles res i d u e s umoles res i d u e s umoles residues umoles r e s i d u e s Lys 0.015 1.00 (1) 0 .080 1.00 (1) His 0 .045 0 .57 (1) 0 .041 0 .70 (1) Arg 0.045 0.90 (1) 0 .045 0 .74 (1) Asp 0.093 1.85 (2) 0.042 2.80 (3) 0 .216 2.70 (3) 0 .123 1.98 (2) MetSC-2 t r a c e Thr 0.017 1.13 (1) 0 .030 0 .83 (1) Ser 0.0 29 1.93 (2) 0 .70 0 .88 (1) Glu 0 .10 5 2.01 (2) 0.014 0.93 (1) 0.068 1.88 (2) 0 .079 1.20 (1) Pro 0.025 1.67 (2) 0.059 1.65 (2) 0 .039 0 .60 (1) Gly 0.160 3.20 (3) 0.035 2.30 (2) 0 .186 2.30 (2) A l a 0.055 1.10 (1) 0 .020 1.30 (1) 0.079 2.19 (2) 0 .189 2.31 (2) V a l 0.155 3.10 (3) 0.018 1.20 (1) 0.041 1.13 (1) 0 .075 0.94 (1) 0 .063 1.03 (1) Met t r a c e (1) Leu 0.047 0.95 (1) 0.014 0 .93 (1) 0 .328 4.10 (4) 0 .055 0.90 (1) T y r t r a c e (1) Phe 0.039 2.62 (3) 0.039 1.08 (1) 0 .066 0.83 (1) 0 .0 82 1.30 (1) corresponding t r y p t i c p e p t i d e I I I V 121 of -130 XIII IX XI Table I I I . Amino acid analyses of t r y p t i c peptides from the peptide map of AE8 (continued) F G H I J * umoles residues ymoles residues ymoles res idues ymoles residues ymoles residues sac** present (1) Lys 0.043 0.92 (1) 0.124 0.96 (1) 0.061 1.90 (2) 0.047 0.98 (1) 0.094 1.30 (1) His 0.098 0.76 (1) 0.027 0.84 (1) 0.041 0 .85 (1) Arg Asp 0.091 2.84 (3) 0.050 1.04 (1) MetS0 2 Thr 0.046 0.98 (1) 0.160 1.23 (1) 0.092 1.91 (2) 0.069 0.95 (1) Ser 0.037 1.15 (1) 0.057 1.19 (1) 0.072 1.00 (1) Glu 0.135 ' 2.87 (3) 0.306 2.30 (2) 0 .061 1.25 (1) Pro 0.084 1.79 (2) 0.139 1.07 (1) Gly 0 .065 2.03 (2) 0.043 0.90 (1) 0.063 0.88 (1) Ala 0.084 1.79 (2) 0.067 2.09 (2) 0.047 0.98 (1) 0.110 1.60 (2) Val 0.061 1.29 (1) 0.101 0.78 (1) 0 .034 1.06 (1) 0.096 1.31 (1) Met Leu 0.156 1.20 (1) 0.129 4.03 (4) 0.089 1.85 (2) 0.076 1.01 (1) Tyr 0.019 0.41 (1) Phe 0.065 1.30 (1) 0 .032 1.00 (1) 0.052 1.08 (1) corresponding t r y p t i c peptide XIII I VIII- IX X II * r v » e i +• i T T O a f a T n t n r f r v n f n n h ftn A m i n n o t h v l eras n Table I I I . Amino a c i d analyses o f t r y p t i c peptides from the peptide map of AEg (continued) K M N 0 ymoles r e s i d u e s ymoles r e s i d u e s ymoles re s i d u e s ymoles re s i d u e s ymoles r e s i d u e s Lys 0.053 1.03 (1) 0.083 1.00 (1) His 0.043 0 .81 (1) 0 .043 1.00 (1) 0.146 1.76 (2) Arg Asp 0.047 0 .89 (1) MetS0 2 Thr Ser Glu Pro Gly 0.067 1.30 (1) 0 .095 1.14 (1) A l a 0 .190 3.73 (4) 0 .113 1.30 (1) V a l 0 .040 1.20 (1) 0.148 2.90 (3) 0 .077 0.93 (1) Met Leu 0 .029 0.89 (1) 0.053 1.04 (1) 0 .073 0.88 (1) T y r 0.021 0 .40 (1) Phe 0 .083 1.00 (1) cor r e s p o n d i n g t r y p t i c 67-68 or pepti d e 113-114 XIV XV XII -b 0 .128 1.07 (1) 0.110 0.92 (1) VI -or A. Table I I I . Amino a c i d analyses o f t r y p t i c peptides from the pe p t i d e map of AE3 (continued) P Q R S T umoles r e s i d u e s umoles r e s i d u e s umoles residues umoles r e s i d u e s umoles r e s i d u e s Lys 0.041 1.05 (1) 0.090 1.12 (1) 0.044 1.00 (1) 0.037 1.70 (2) His 0.060 0.75 (1) 0.025 1.13 (1) Arg 0.065 0.9 8 (1) Asp MetSC-2 Thr 0.060 0.92 (1) Ser 0.075 1.15 (1) Glu 0 .038 0.97 (1) Pro Gly 0.102 1.15 (1) 0.030 1.30 (1) A l a 0.103 1.17 (1) 0.023 1.04 (1) V a l Met Leu T y r Phe corresponding t r y p t i c 38-40 131-132 VII V I I I V I I - V I I I p e p t i d e • •. -CO to 83 p e p t i d e s gave a p o s i t i v e c o l o r w i t h the s p e c i f i c s t a i n p r i o r t o i s o l a t i o n ( F i g . 12). The amino a c i d analyses of B, E and L p e p t i d e s confirmed the s t a i n i n g data and c o u l d be equated w i t h p e p t i d e s BTpV, XI and XIV r e s p e c t i v e l y . In summary, the amino a c i d composition of G,J,A,B,0,R,S,T,D,I,E,N,F,L and M r e p r e s e n t e d the t r y p t i c p e p t i d e number I, I I , I I I , V, VI, V I I , V I I I , V I I - V I I I , IX, X, XI, X l l - b , X I I I , XIV and XV o f B-chain r e s p e c t i v e l y (Table I I I , F i g . 12, 12a and 13). The amino a c i d analyses of the C, Q, K and P appeared to correspond to the fragments: r e s i d u e s 121-130, Glu-Phe-Thr-Pro-Pro-Val-Gln-Ala-Ala-Tyr; r e s i d u e s 131-132, Gln-Lys; r e s i d u e s 67-68 o r 113-114, Va l - L e u and r e s i d u e s 38-40, Thr-Gln-Arg r e s p e c t i v e l y (Table I I I , F i g . 12 and 13). The presence of these fragments, suggests t h a t the t r y p s i n used was contaminated w i t h chymotrypsin g i v i n g r i s e t o C - t e r m i n a l cleavages a t the 130 t y r o s y l , 6 8 o r 114 l e u c y l and 38 t r y p t o p h a n y l r e s i d u e s o f the t r y p t i c p e p t i d e s . Consequently p e p t i d e s C,Q,K and P a r i s e (see F i g . 13). A c c o r d i n g to s p e c i a l s t a i n i n g , the W p e p t i d e corresponds to BTpIV ( F i g . 12 and 13X, but i n s t e a d o f b e i n g a major p e p t i d e i t appeared only a f a i n t o r minor p e p t i d e on the map ( P l a t e 11, F i g . 12). T h i s observ-a t i o n can be e x p l a i n e d i f the m a j o r i t y o f BTpIV was h y d r o l y z e d by the chymotrypsin, at l e a s t a t the C - t e r m i n a l of 38 t r y p t o p h a n y l r e s i d u e . The BTpXII-a (see F i g . 13), expected as a major pe p t i d e o f t r y p t i c AEB map, was a l s o m i s s i n g but i t a l s o may have been h y d r o l y z e d by the a c t i o n of chymotrypsin a t the c a r b o x y l end o f l e u c y l r e s i d u e s . 84 C. Summary A A T r y p t i c p e p t i d e s of non s u b s t i t u t e d ct and AEg on p e p t i d e maps u s i n g HVE pH 6.5 and n - b u t a n o l - a c e t i c a c i d - w a t e r - p y r i d i n e system (120) were i d e n t i f i e d b a s i n g on s p e c i a l s t a i n i n g and amino a c i d a n a l y s e s . The r e s o l u t i o n o f both t r y p t i c p e p t i d e s b e i n g summarized on F i g . 9a, and F i g . 12a were used as r e f e r e n c e s f o r the f o l l o w i n g s t u d i e s on the s t r u c t u r a l c h a r a c t e r i z a t i o n o f abnormal hemoglobins. 85 V. Hb j B a n 9 k o k I N A CHINESE NEWBORN IN VANCOUVER, B.C. A f a s t abnormal hemoglobin was d e t e c t e d on s t a r c h g e l e l e c t r o p h o r e s i s with t r i s - E D T A - b o r a t e b u f f e r , pH 8.6, i n a cord b l o o d d u r i n g a survey o f Hb B a r t ' s i n Chinese Canadian newborns i n Vancouver, B. C , by Gray and Marion (141) . The f a m i l y study and h e m a t o l o g i c a l i n v e s t i g a t i o n s were c a r r i e d out a t the D i v i s i o n o f Hematology, Department of Pathology, V.G.H. (141) . A. C l i n i c a l S t u d i e s 1. Family study The p r o p o s i t u s was female, a f u l l term d e l i v e r y o f 6 l b . 15 oz. and p h y s i c a l examination showed no a b n o r m a l i t i e s . A f a m i l y study r e v e a l e d t h a t the f a t h e r appeared h e a l t h y and a l s possessed the abnormal f a s t hemoglobin while the mother and one b r o t h e r were normal. The p a t e r n a l grandparents o r i g i n a t e d in^Canton r e g i o n of China. The pedigree i s shown on F i g . 14. 2. He m a t o l o g i c a l i n v e s t i g a t i o n s The f a s t abnormal hemoglobin was found i n the c o r d b l o o d the p r o p o s i t u s t o the ext e n t o f 17% i n a d d i t i o n t o Hb A and Hb F. Her parents and one b r o t h e r were examined. The f a t h e r I I - l (see F i g . 14) showed 54% o f abnormal pigment w i t h 2.2% Hb A 2 and 0.7% a l k a l i - r e s i s t a n t hemoglobin. The h e m a t o l o g i c a l f i n d i n g s i n both heterozygotes were normal. 86 r - i i 0 i J2 0 I i i /3 (J^ ) Normol X Propositus r 1 I J Not tested Dead F i g u r e 14. The p e d i g r e e o f H b J B a n g k o k f a m i l y 87 B. B i o c h e m i c a l I n v e s t i g a t i o n In order t o c h a r a c t e r i z e the mo l e c u l a r pathology o f t h i s abnormal pigment, a hemolysate from I I - l was b i o c h e m i c a l l y i n v e s t i g a t e d . 1. Hemoglobin e l e c t r o p h o r e s i s and hemoglobin c h a i n d i s s o c i a t i o n study The hemoglobin s t a r c h g e l e l e c t r o p h o r e s i s w i t h t r i s - E D T A -b orate b u f f e r , pH 8.6 showed a l a r g e amount of f a s t abnormal hemoglobin (over 50% o f t o t a l hemoglobin) ( P l a t e 15). F or l o c a t i o n o f the a l t e r e d g l o b i n c h a i n o f the v a r i a n t , the hemo-g l o b i n chains were d i s s o c i a t e d by treatment w i t h PHMB and the products were examined by s t a r c h g e l e l e c t r o p h o r e s i s i n the presence o f PHMB (56). An abnormal g - c h a i n which moved r a p i d l y towards the anode c o u l d be seen ( P l a t e 16). T h i s i n d i c a t e d t h a t the v a r i a n t g - c h a i n must possess a d d i t i o n a l negative charges. 2. I s o l a t i o n o f the abnormal g l o b i n c h a i n G l o b i n , prepared from the hemolysate by a c i d acetone p r e c i p i t a t i o n was f r a c t i o n a t e d by C M - c e l l u l o s e chromatography (74). The chromatogram showed a f a s t abnormal g - c h a i n which comprised more than 50% o f t o t a l g - c h a i n ( F i g . 17). Both abnormal and normal g-chairewere aminoethylated and d e s a l t e d on Sephadex G-25. The aminoethylated g - c h a i n o f both v a r i a n t and normal were d i g e s t e d w i t h TPCK-trypsin at the enzyme s u b s t r a t e r a t i o 1/100 f o r 2 1/2 hours. 88 P l a t e 15. Hemoglobin s t a r c h g e l e l e c t r o p h o r e s i s , t r i s - E D T A -b o r a t e b u f f e r , pH 8.6. 89 P l a t e 16. Hemoglobin c h a i n d i s s o c i a t i o n w i t h PHMB on s t a r c h g e l e l e c t r o p h o r e s i s w i t h t r i s - E D T A - b o r a t e b u f f e r pH 8.6. F i g u r e 17. C M - c e l l u l o s e chromatography o f 30 mg o f g l o b i n i n 8M u r e a and 0.05M m e r c a p t o e t h a n o l w i t h sodium phosphate b u f f e r pH 6.7. A m i x t u r e o f s t a r t i n g and c o n c e n t r a t e d b u f f e r (200 ml o f each) were mixed t o g i v e a l i n e a r g r a d i e n t . 91 3. L o c a t i o n of abnormal t r y p t i c p e p t i d e on pept i d e map Comparative t r y p t i c d i g e s t mapping o f normal and abnormal 8-chain were performed by h i g h v o l t a g e e l e c t r o p h o r e s i s pH 6.5 and descending chromatography. The combination o f s t a i n s ; c h l o r o p l a t i n a t e , 0.2% n i n h y d r i n and Pauly s t a i n s were employed. The r e s u l t s showed t h a t a methionine c o n t a i n i n g p e p t i d e i n the abnormal 8-chain was d i s p l a c e d towards the anode when compared with the co r r e s p o n d i n g m e t h i o n i n e - p o s i t i v e p e p t i d e B^TpV ( t r y p t i c peptide No. 5 from N - t e r m i n a l of 8 - c h a i n ) ( P l a t e 18). Another spot which showed the same e l e c t r o p h o r e t i c m o b i l i t y as the methionine p e p t i d e had a s l i g h t l y slower m i g r a t i o n when paper chromatographed and d i d not s t a i n f o r methionine. T h i s i s thought to be the o x i d i z e d form of the methionine peptide (74,142). These f i n d i n g s i n d i c a t e d t h a t the a l t e r a t i o n which i s r e s p o n s i b l e f o r the i n c r e a s e d n e g a t i v e charge of the abnormal 8-chain i s l o c a t e d i n BTpV. 4. Amino a c i d a l t e r a t i o n i n BTpV o f the v a r i a n t In o r d e r t o determine the amino a c i d s u b s t i t u t i o n i n the mutant, comparative amino acid., analyses of the abnormal gTpV and B^TpV were performed. A t r y p t i c p e p t i d e map o b t a i n e d w i t h a 5 mg of normal 8-chaxn (3 ) was s t a i n e d v/ith 0.02% n i n h y d r i n , the corresponding methionine p e p t i d e l o c a t e d and then e l u t e d w i t h 6N HCl. The e l u a t e was h y d r o l y z e d a t 110° C f o r 24 hours under vacuum. A f t e r d r y i n g the h y d r o l y s a t e was a p p l i e d t o the a n a l y z e r . The r e s u l t of amino a c i d a n a l y s i s of the normal pept i d e confirmed the known amino a c i d composition o f 8 TpV 92 93 but the g v a r i a n t p e p t i d e , t r e a t e d s i m i l a r l y , showed one l e s s g l y c i n e r e s i d u e and an a d d i t i o n a l r e s i d u e of a s p a r t i c a c i d (Table I V ) . The i n c r e a s e i n n e g a t i v e charge of the mutant g-chain i n d i c a t e s t h a t g l y c i n e i s s u b s t i t u t e d by a s p a r t i c a c i d r a t h e r than asparagine s i n c e the l a t t e r would not cause the a l t e r a t i o n i n m o b i l i t y seen upon e l e c t r o p h o r e s i s a t pH 6.5 and C M - c e l l u l o s e chromatography. The replacement of g l y c i n e by a s p a r t i c a c i d might occur e i t h e r a t g l y c i n e r e s i d u e s 46 or 56 of gTpV (see F i g . 19) . 5. L o c a t i o n o f amino a c i d a l t e r a t i o n i n the CNBr p e p t i d e , between p o s i t i o n s 56-59 o f gTpV Upon high v o l t a g e e l e c t r o p h o r e s i s a t pH 6.5, the abnormal p e p t i d e gTpV ran s l i g h t l y f a s t e r and the normal g^TpV s l i g h t l y slower than the b l u e dye marker XCFF (xylene c y a n o l blue F F ) . The m o b i l i t y of XCFF was, t h e r e f o r e , a guide to the progress of a prolonged 3 hours h i g h v o l t a g e run f o r the p r e p a r a t i o n of the two p e p t i d e s which were f i n a l l y l o c a t e d by the c h l o r o p l a t i n a t e s t a i n f o r methionine. Both p e p t i d e s were e l u t e d from u n s t a i n e d papers w i t h d i l u t e NH^OH and the s o l u t i o n l y o p h i l i z e d . Each pept i d e was then c l e a v e d w i t h cyanogen bromide and a s i x t h p o r t i o n was r e - r u n i n HVE a t pH 6.5. I t may be seen from P l a t e 20 t h a t t h r e e n i n h y d r i n - p o s i t i v e , methionine-negative spots were obt a i n e d . CNBrI i n each case showed the same m o b i l i t y i n r e l a t i o n to XCFF as the o r i g i n a l p e p t i d e and probably r e p r e s e n t s i t s methionine s u l f o x i d e form which i s r e s i s t a n t t o CNBr cleavage. 94 Ta b l e IV. Amino a c i d compositions o f methionine p o s i t i v e p e p t i d e (BTpV) of normal and abnormal B-chains B ^ p V - 3 JTpV ymoles Residues ymoles : Residues Lys 0.005 0.57 0 .017 0.97(1) His Arg Asp 0.027 2.87(3) 0.072 4.07(4) Met s u l f o n e * 0.00 3 0.34 Trace Thr 0.009 0.98(1) 0.018 1.0 (1) Ser 0.018 1.95(2) 0 .036 2.03(2) Glu 0.010 1.06(1) 0 .021 1.2 (1) Pro 0.007 0.76(1) 0.022 1.2 (1) Gly 0.021 2.26(2) 0.025 1.3 (1) A l a 0.009 0.99(1) 0.024 1.1 (1) V a l 0.006 0.69(1) 0.015 0.87(1) Meth Trace (1) Trace CD H e Leu 0 .009 0.96(1) 0.018 1.02(1) Tyr Phe 0.024 2.53(3) 0.050 2.84(3) 5 mg of sample = 0. 30 3 ymoles •Methionine s u l f o n e was a r e s u l t o f o x i d a t i o n o f methionine Normal 0 ATpV 41 46 55 56 59 Phe-Phe-Glu-Ser-Phe-Gly-Asp-Leu-Ser-Thr-Pro-Asp-Ala-Val-Met-Gly-Asn-Pro-Lys CNBr II-A — C N B r III-A->" CNBr I-A (oxidized) Abnormal B JTpV 41 46 55 56 59 Phe-Phe-Glu-Ser-Phe-Gly-Asp-Leu-Ser-Thr-Pro-Asp-Ala-Val-Met-Asp-Asn-Pro-Lys CNBr I I - J *-|«—CNBr I I I - J - * -CNBr I - J (oxidized) • * A J F i g u r e 19. Diagram of the sequence o f 3 TpV and 3 TpV t r e a t e d w i t h cyanogen bromide, 97 C N B r l l - A and C N B r l l l - A p e p t i d e s from 6 TpV which were a c i d i c and b a s i c r e s p e c t i v e l y correspond t o the p e p t i d e 41-55, Phe-Phe-Glu-Ser-Phe-Gly-Asp-Leu-Ser-Thr-Pro-Asp-Ala-Val-Hs1* and 56-59, Gly-Asn-Pro-Lys, expected by cleavage at the C - t e r m i n a l s i d e o f the m e t h i o n y l r e s i d u e 55 (see F i g . 19). In the case o f the abnormal p e p t i d e , the a c i d i c p e p t i d e C N B r l l - J was i d e n t i c a l i n m o b i l i t y t o C N B r l l - A from 8 TpV i n d i c a t i n g t h a t p o s i t i o n 41-55 were unchanged. However, C N B r l l l - J from the v a r i a n t , i n s t e a d of b e i n g b a s i c , was now n e u t r a l w i t h the m o b i l i t y e q u a l t o e-DNP-lysine. Thus the s i t e of a l t e r a t i o n must be i n t h i s p e p t i d e l o c a t e d between p o s i t i o n s 56-59 of 8TpV. 6. The s i t e o f mutation i n the 8-chain i s g l y c i n e r e s i d u e 56 which i s r e p l a c e d by a s p a r t i c a c i d . The remaining p o r t i o n o f cyanogen bromide t r e a t e d BTpV of normal and v a r i a n t were f r a c t i o n a t e d by h i g h v o l t a g e paper e l e c t r o p h o r e s i s , pH 6.5, a t 3 KV f o r 1 1/2 hours. A f t e r l o c a t i o n of the p e p t i d e s by cadmium-ninhydrin s t a i n i n g on a guide s t r i p , the c o r r e s p o n d i n g papers of t h r e e u n s t a i n e d CNBr p e p t i d e s were e l u t e d w i t h d i l u t e NHi»OH and l y o p h i l i z e d . The p e p t i d e s were h y d r o l y z e d w i t h c o n s t a n t b o i l i n g 5.7 N HCl a t 110° C f o r 24 hours under vacuum. T h e i r amino a c i d analyses were c a r r i e d out and are shown on T a b l e V. As expected, the a n a l y s i s of the CNBrl-A and CNBrl-J proved t o be the o x i d i z e d form o f the methionyl p e p t i d e of B ^ p V and mutant BTpV r e s p e c t i v e l y . *Hsl=homoserdne l a c t o n e T a b l e V. Amino A c i d C o m p o s i t i o n s o f CNBr C l e a v a g e o f B ^ p V and B JTpV CNBr-B^pV CNBr-3 JTpV I**-A I I - A I I I - A I * * * - J I I - J ~" I I I - J y m o l e R e s i d u e ymole Residue ymole Residue ymole R e s i d u e ymole Residue ymole Residue Lys 0 .007 1. 0 (1) 0 .00 4 0 .80(1) 0 .009 0.98(1) 0 .032 1 .0 (1) His Arg - - • j Asp 0 .022 3. 1 (3) 0.024 2.1 (2) 0 .007 1 .3 (1) 0.039 4.1 (4) 0 .026 1 .91(2) 0 .064 2 .01(2) Met SO* 0 .002 0 . 34(1) t r a c e (1) Thr 0 .00 7 1. 0 (1) 0.011 0.97(1) 0.009 0.90(1) 0 .012 0 .86(1) -Ser 0 .013 1. 91(2) 0.023 2.01(2) 0.018 1.93(2) 0 .024 1 .79(2) 0 .003 0 .09 (0) Hsr 0.008 0.68(1) 0 .00 8 0 .6 (1) — Glu 0 .009 1. 23(1) 0.012 1.02(1) 0.010 1.07(1) 0 .015 1 .12(1) 0 .001 0 .02(0) Pro 0 .012 1. 78(2) 0.007 0.57(1) 0 .003 0 .62(1) 0 .011 1.17(1) 0 .008 0 .57(1) 0 .025 0 .81(1) Gly 0 .015 2. 12(2) 0.012 1.03(1) 0 .006 1 .19(1) 0.012 1.3 (1) 0.015 1 .09(1) 0 0 A l a 0 .007 0. 99(1) 0.011 0.96(1) 0.009 0 .92 (1) 0 .018 1 .30(1) V a l 0 .007 1. 03(1) 0 .014 1.22(1) 0 .007 0.74(1) 0 .009 0 .67(1) Meth -l i e u Leu 0 .007 1. 01(1) 0 .012 1.01(1) 0 .00 8 0.89(1) 0 .013 0 .96(1) Tyr Phe 0 .018 2. 52(3) 0 .031 2.63(3) 0.033 3.4 (3) 0 .044 3 .2 (3) vo cn T a b l e V. c o n t i n u e d . . . * M e t h i o n i n e s u l f o n e was a r e s u l t o f o x i d a t i o n o f M e t h i o n i n e . ** O x i d i z e d g ATpV *** O x i d i z e d B JTpV H s r = Homoserine a n a l y s i s VO VD 100 The C N B r l l - A p e p t i d e was i d e n t i c a l t o C N B r l l - J c o r r e s p o n d i n g t o r e s i d u e s 41-55 of the 3-chain. However, the amino a c i d a n a l y s i s o f C N B r l l l - J , supposing to correspond to C N B r l l l - A which r e p r e s e n t s r e s i d u e s 56-59 o f the 8-chain (Table V) gave no g l y c i n e and two a s p a r t i c a c i d r e s i d u e s i n d i c a t i n g t h a t the g l y c i n e a t p o s i t i o n 56 was s u b s t i t u t e d by a s p a r t i c a c i d . 7. C o n f i r m a t o r y evidence f o r the s i t e of mutation As i t i s known from the sequence ( F i g . 19) the g l y c i n y l r e s i d u e 56 i s l o c a t e d next t o methionine r e s i d u e 55 of the 8-chain. Thus the cyanogen bromide cleavage of B^TpV should g i v e a phenyl a l a n i n e and g l y c i n e as the N - t e r m i n i of C N B r l l - A and C N B r l l l - A r e s p e c t i v e l y . However, f o r the mutant i n which the g l y c i n e a t r e s i d u e 56 i s r e p l a c e d by a s p a r t i c a c i d , s i m i l a r treatment, would g i v e p h e n y l a l a n i n e and a s p a r t i c a c i d as N - t e r m i n i of C N B r l l - J , and C N B r l l l - J r e s p e c t i v e l y (see F i g . 1 9 ) . Based on t h i s r e a s o n i n g , the s t u d i e s of the N - t e r m i n i o f t o t a l u n f r a c t i o n a t e d CNBr r e a c t i o n mixtures of normal and abnormal BTpV were compared. D a n s y l a t i o n (132) and i d e n t i f i c a t i o n o f DNS-amino a c i d s by t h i n l a y e r chromatography (133) a f t e r a c i d h y d r o l y s i s showed t h a t DNS-phenylalanine and DNS-glycine were rec o v e r e d from 8 TpV w h i l e the abnormal p e p t i d e gave DNS-phenyalanine and DNS-aspartic a c i d as expected ( P l a t e 2 1 ) . C. D i s c u s s i o n A g e n e r a l summary o f these data with the sequences of the normal and abnormal BTpV i s given i n F i g . 19 and leads to the c o n c l u s i o n t h a t the abnormal hemoglobin observed i n t h i s p a t i e n t 101 • * • • • Apolar solvent system Polar solvent system -DNS-As? Origin P l a t e 21. T h i n l a y e r chromatography o f DNS-amino a c i d s (see t e x t s ) 1,16 = V a l ; 2,8,12 = G l y ; 3,7,11 = Asp; 4*9,13 = u n f r a c t i o n a t e d CNBr r e a c t i o n m i x t u r e o f 3 TpV; 5,10,14 = u n f r a c t i o n a t e d CNBr m i x t u r e o f 8 TpV. 102 i s due t o an amino a c i d s u b s t i t u t i o n o f g l y c i n e f o r a s p a r t i c a c i d a t p o s i t i o n 56 of the.3-ch.ain. T h i s mutation has been observed p r e v i o u s l y as H b J B a n g k o k which was f i r s t d e s c r i b e d i n a h e a l t h y newborn of a Thai f a m i l y by P o o t r a k u l , Wasi and Na-Nakorn (73), subsequent b i o c h e m i c a l i n v e s t i g a t i o n showed t h i s mutant to be a2852 6 G l y " * A s p (74) . Other f a s t hemoglobin, desi g n a t e d as H b J K o r a t i n a T h a i f a m i l y (143) and H b J M e i n u n g among Hakkanese Chinese i n Taiwan (144) were a l s o b e l i e v e d t o show the same mutation (145). The pr e s e n t o b s e r v a t i o n of H b J B a n g k o k i n a Chinese f a m i l y i n Vancouver, B.C., o r i g i n a t i n g i n the Canton area o f South China, i s c o n s i s t e n t with the occurrence and p r e v i o u s o b s e r v a t i o n o f t h i s mutation i n South-East A s i a . Furthermore, t h i s v a r i a n t which i s a r a r e abnormal hemoglobin, has been so f a r d e s c r i b e d o n l y among T h a i (73,143) and Chinese f a m i l i e s (144,145) and can probably be c o n s i d e r e d as a g e n e t i c marker f o r T h a i and Chinese p o p u l a t i o n s . The g l y c y l r e s i d u e at p o s i t i o n 56 of the 8-chain i s l o c a t e d on the o u t s i d e o f hemoglobin molecule a t D7 (r e s i d u e 7 of h e l i c a l segment D)(49,77) and thus i t s mutation t o a s p a r t i c a c i d , a h i g h l y p o l a r r e s i d u e which can i n t e r a c t w i t h the s o l v e n t w i t h o u t causing any c o n f o r m a t i o n a l change, i s c o n s i s t e n t w i t h the u n a l t e r e d s t a b i l i t y and normal c l i n i c a l f i n d i n g s of H b J B a n g k ° k . 10 3 D. A Summary A f a s t ( a n i o n i c ) abnormal hemoglobin was d e t e c t e d i n c o r d b l o o d o f a Chinese baby, o f Cantonese descent, d u r i n g a hemoglobin B a r t ' s survey i n newborns a t Vancouver General H o s p i t a l , B. C. The heterozygotes p o s s e s s i n g t h i s v a r i a n t , the p r o p o s i t u s and h e r f a t h e r , showed normal h e m a t o l o g i c a l f i n d i n g s except f o r the presence o f abnormal pigment. From b i o c h e m i c a l s t u d i e s , i t i s e v i d e n t t h a t the g l y c i n e a t r e s i d u e 56 i n 6-chain i s s u b s t i t u t e d by a s p a r t i c a c i d . T h i s mutation i s i d e n t i c a l t o t h a t p r e v i o u s l y d e s i g n a t e d as H b J B a n g k o k ( a A e ! 6 A s p ) . 10 4 VI. A DOUBLE HETEROZYGOTE OF HbE AND Hb NEW YORK IN A THAI FAMILY A slow hemoglobin and f a s t hemoglobin i n the absence of HbA was d e t e c t e d on a s t a r c h g e l e l e c t r o p h o r e s i s with t r i s -EDTA-borate b u f f e r pH 8.6 from an hemolysate d u r i n g the r o u t i n e examination o f hemoglobin type o f a new p a t i e n t a t t e n d i n g the Hematology C l i n i c , S i r i r a j H o s p i t a l , Bangkok. The slow hemoglobin was b e l i e v e d t o be HbE on the b a s i s o f the m o b i l i t y on s t a r c h g e l e l e c t r o p h o r e s i s and high i n c i d e n c e o f t h i s mutant among Tha i p o p u l a t i o n s (146,147,148). The f a s t abnormal hemoglobin migrated s l i g h t l y ahead o f the HbA c o n t r o l but slower than Hb B a r t ' s . The c l i n i c a l , h e m a t o l o g i c a l and g e n e t i c a l s t u d i e s on the abnormal hemoglobins were c a r r i e d out by the D i v i s i o n o f Hematology, Department of Medicine, Mahidol U n i v e r s i t y , Bangkok. A. C l i n i c a l I n v e s t i g a t i o n s 1. G e n e t i c a l study The p r o p o s i t u s , a 22 yea r o l d man of T h a i descent, came t o the Hematology C l i n i c f o r a r o u t i n e b l o o d examination due t o p y r e x i a . The c l i n i c a l f i n d i n g s r e v e a l e d no a b n o r m a l i t i e s and the u s u a l h e m a t o l o g i c a l i n v e s t i g a t i o n s i n c l u d i n g hemoglobin type were performed. A f t e r the d e t e c t i o n o f the abnormal hemoglobins, f a m i l y s t u d i e s were c a r r i e d out i n 8 r e l a t i v e s ( F i g . 22). I t can be seen t h a t h i s f a t h e r , ';iI-3 possessed the f a s t abnormal hemoglobin which e v i d e n t l y was t r a n s f e r r e d from h i s g r a n d f a t h e r I - l , w h i l e h i s mother II-4 i n h e r i t e d the slow hemoglobin - HbE. 105 F i g u r e 22. Pedigree of a double heterozygote of HbE and Hb New York i n a Thai f a m i l y . 106 Furthermore, one of h i s b r o t h e r s I I I - 4 was a l s o found t o be a double heterozygote o f the slow and f a s t Hb and l a c k i n g HbA. The heterozygote o f HbE, I I I - 3 and f a s t Hb i n I I I - 2 , I I I - 5 were a l s o observed. The heterozygote o f HbE or f a s t Hb and the double heterozygote of both v a r i a n t s as w e l l , appeared h e a l t h y . 2. Hem a t o l o g i c a l f i n d i n g s A summary of the h e m a t o l o g i c a l f i n d i n g s i s shown on Tab l e V I . The heterozygotes f o r the f a s t abnormal hemoglobin, I - l , I I - 3 , I I I - 2 and I I I - 5 as w e l l as E t r a i t , I I - 4 , I I I - 3 , a p p a r e n t l y Showed normal h e m a t o l o g i c a l f i n d i n g s b e s i d e s the presence of the abnormal pigment. U n f o r t u n a t e l y the q u a n t i t y of the f a s t abnormal hemoglobin i n t r a i t form was not determined but i n the heterozygote HbE, the abnormal pigment was 27.5 and 26.5% of t o t a l hemoglobin f o r II-4 and I I I - 3 r e s p e c t i v e l y . The double h e t e r o z y g o t e , I I I - l and III - 4 r e v e a l e d no h e m a t o l o g i c a l abnorm-a l i t i e s except f o r the p a t t e r n of hemoglobin e l e c t r o p h o r e s i s . The amount of HbE was 33.2 and 32.9% w h i l e the measurement of a l k a l i d e n a t u r a t i o n o f the hemoglobin was 1.6 and 0.8% f o r I I I - l and III - 4 r e s p e c t i v e l y . The r e s t of the hemoglobin was assumed to be the f a s t abnormal pigment. B. B i o c h e m i c a l I n v e s t i g a t i o n s A b l o o d sample was o b t a i n e d from p r o p o s i t u s , I I I - l . A f t e r washing with 0.9% NaCl f o r three times the packed red c e l l s were f r o z e n i n d r y - i c e f o r t r a n s p o r t a t i o n t o Vancouver. The hemolysate was prepared by the water toluene method. Table VI. A Summary of Hematological F i n d i n g s and Desi g n a t i o n s 'edigree Hb RBC PCV R e t i c RBC Osmotic SI/UIBC Morph o logy Fr a g i l i t y Hb Type Hb A 2 o r E A l k a l i Denat. D e s i g n a t i o n : - i 12.3 4.57 41 1.2 N N - A+NY A 2 - NY t r a i t :-2 12.9 4.67 41 0 N N 58/208 A+A2 1.19 0.2 Normal :i-3 15.5 4.76 45 1.2 N s l i g h t l y i n c r e a s e d - A+NY A 2 2.93 0.7 NY t r a i t :i-4 13.8 5.55 41 1.6 N N 147/137 A+E 27.5 0.3 E t r a i t : n - i 14.6 5.69 46 2.8 N N 77/150 E+NY 33.2 1.6 Double h e t e r o z y -gote E+NY [II-2 13.8 4.8 44 0.4 N N 60/259 A+NY A 2 2.29 1.8 NY t r a i t [II-3 13.4 4.64 43 0.4 N N 83/384 E+A 26.5 - E t r a i t EII-4 15.4 5.96 46 1.6 N - N 115/195 E+NY 32.9 0.8 Double h e t e r o z y -gote E+NY CII-5 16.8 5.97 50 0.8 N N 154/- A+NY +A2 2.6 0.4 NY t r a i t N = normal, SI = serum i r o n , UIBC = unsaturated i r o n b i n d i n g c a p a c i t y 10 8 1. Hemoglobin e l e c t r o p h o r e s i s The hemolysate was s u b j e c t e d t o a s t a r c h g e l e l e c t r o p h o r e s i s with t r i s - E D T A - b o r a t e b u f f e r , pH 8.6 (P l a t e 23). S i m i l a r l y t o the p r e v i o u s f i n d i n g i n Bangkok, the sample I I I - l showed only two major components namely a slow hemoglobin - b e l i e v e d t o be HbE and a f a s t hemoglobin which moved j u s t ahead of the HbA c o n t r o l towards the anode. From the s t a r c h g e l i t may be seen t h a t no HbA was observed and the amount of HbE appeared t o be l e s s than the f a s t hemoglobin. T h i s was c o n s i s t e n t with the q u a n t i t a t i v e d e t e r m i n a t i o n . In the absence o f HbA, the HbE -a slow 8 mutant c o u l d be i n v o l v e d i n one o f two p o s s i b l e i n t e r a c t i o n s namely with a 8-thalassemia o r with another 8-chain v a r i a n t . In t h i s case, the former p o s s i b i l i t y was r u l e d out f o r a number o f reasons w h i l e the l a t t e r was c o n s i d e r e d most l i k e l y . The e v i d e n c e , t h e r e f o r e , suggested a double h e t e r -ozygote o f a slow 8 mutant - HbE and a f a s t 8-chain v a r i a n t . 2. To demonstrate the mutant g l o b i n c h a i n The hemoglobin was d i s s o c i a t e d i n t o g l o b i n chains by the treatment o f PHMB (56) and the product was s u b j e c t e d t o a s t a r c h g e l e l e c t r o p h o r e s i s i n the presence o f PHMB (P l a t e 24). While no 8 was seen t h r e e other g l o b i n chains were observed namely: a f a s t abnormal 8-chain; another g l o b i n c h a i n mutant -A A A m i g r a t i n g between 8 and a ; and normal a . The f a s t abnormal 8 g l o b i n c h a i n was b e l i e v e d t o d i s s o c i a t e from the f a s t abnormal hemoglobin. The oth e r abnormal g l o b i n c h a i n , moving between A A a and 8 might be a f a s t a or slow 8 mutant, however, t h i s 109 O r i g i n P r opositus (II I - I ) Heterozygote of Hb Mahidol Normal hemolysate I Hb New York I Anode HbA2 or HbE HbA P l a t e 23. Hemoglobin s t a r c h g e l e l e c t r o p h o r e s i s o f a double heterozygote of a slow hemoglobin - HbE and a f a s t hemoglobin - Hb New York, from the p r o p o s i t u s I I I - l sample i n t r i s - E D T A - b o r a t e b u f f e r pH 8.6. 110 P l a t e 24. S t a r c h g e l e l e c t r o p h o r e s i s i n t r i s - E D T A - b o r a t e b u f f e r pH 8.6 o f hemoglobin c h a i n d i s s o c i a t e d by PHMB t r e a t m e n t . I l l abnormal g l o b i n c h a i n must have a r i s e n by d i s s o c i a t i o n from the other slow 3 mutant - HbE. Thus t h i s abnormal g l o b i n c h a i n was the slow 8 mutant - 8 which was c o n s i s t e n t w i t h the pr e v i o u s A^  hemoglobin e l e c t r o p h o r e s i s and the absence o f 3 a f t e r hemoglobin c h a i n d i s s o c i a t i o n . In summary, a study o f hemoglobin d i s s o c -i a t i o n w i t h PHMB confirmed the double heterozygote o f a f a s t 8 - c h a i n mutant and a slow 8-chain v a r i a n t o f 8 as p r e v i o u s l y s p e c u l a t e d . 3. To i s o l a t e both slow and f a s t 8 g l o b i n c h a i n mutants 250 mg of g l o b i n , prepared from a hemolysate by a c i d -acetone p r e c i p i t a t i o n , was chromatographed on a 2.5x18 cm column of C M - c e l l u l o s e c o n t a i n i n g 8M urea, 0.05M 8-mercaptoethanol and sodium phosphate b u f f e r , pH 6.7 (74). The l i n e a r g r a d i e n t o f sodium ions was used by mixing equal volumes (1400 mis) o f 0.00 5M s t a r t i n g b u f f e r and 0.03M c o n c e n t r a t e d b u f f e r , 15 mis per f r a c t i o n were c o l l e c t e d . The chromatograph i s shown on NY E A F i g . 25. Three major chains were obtained; 8 , 8 and ct . A The 8 c h a i n which u s u a l l y e l u t e s a t 1.0-1.2 m i l l i mho i n the g r a d i e n t was not observed, w h i l e 8 and 6 f r a c t i o n a t e d a t a lower and h i g h e r i o n i c s t r e n g t h than would be expected f o r 8 A NY E t h e r e f o r e , 8 i s more i o n i c and 8 - l e s s i o n i c which i s c o n s i s t e n t w i t h the PHMB g l o b i n c h a i n d i s s o c i a t i o n study. The aminoethylated g l o b i n c h a i n o f both v a r i a n t s were prepared (74) and d i g e s t e d w i t h T P C K - t r y p s i n f o r 2 1/2 hours a t the enzyme s u b s t r a t e r a t i o n 1 t o 100. The aminoethylated chains were l y o p h i l i z e d . F i g u r e 25. G l o b i n c h a i n f r a c t i o n a t i o n o f I I I - l sample by C M - c e l l u l o s e chromatography (see t e x t ) . 113 4. To c o n f i r m the i d e n t i t y o f the slow 8 v a r i a n t as being HbE ( a 2 8 i e G l u " L Y S ) E The t r y p t i c p e p t i d e map o f AEB was c a r r i e d out by HVE pH 6.5 f o l l o w e d by descending chromatography (120) and compared A, w i t h a p e p t i d e map o f AEB , The combination o f n i n h y d r i n and Sakaguchi s t a i n f o r a r g i n i n e was employed. The chromatograras are shown on P l a t e 26. By comparison w i t h a normal p e p t i d e map A of AEB ( F i g . 12a, and P l a t e 26) i t i s e v i d e n t t h a t the a r g i n y l p e p t i d e c o r r e s p o n d i n g to B T p I I l i n the v a r i a n t i s m i s s i n g w h i l e E two new major p e p t i d e s ; namely B T p I I I - a , a s l i g h t l y a c i d i c , E a r g i n i n e - n e g a t i v e p e p t i d e and 8 TpIH-b a b a s i c , a r g i n i n e c o n t a i n i n g p e p t i d e appeared. Since under these c o n d i t i o n s t r y p s i n c l e a v e s only a t the p e p t i d e bonds c o n t a i n i n g l y s i n e and a r g i n i n e , the amino a c i d s u b s t i t u t i o n must be l y s i n e or a r g i n i n e . Supposing a r g i n i n e were the mutation, both new p e p t i d e s would be expected to g i v e a p o s i t i v e o f Sakaguchi s t a i n . But i n E t h i s case o n l y b a s i c p e p t i d e 8 TpIII-b d i d , t h e r e f o r e the a l t e r a t i o n t o l y s i n e was s t r o n g l y suggested. A f t e r s t a i n i n g w i t h 0.02% n i n h y d r i n , the p e p t i d e s corresponding E E to 8 T p I I I - a and 8 TpIII-b o b t a i n e d from a p e p t i d e map of 5 mg E o f t r y p t i c AEB were c u t out and e l u t e d w i t h 6N HCl. The e l u a t e s were h y d r o l y z e d a t 110° C f o r 24 hours under vacuum and h y d r o l y s a t e s s u b j e c t e d t o amino a c i d a n a l y s i s (Table V I I ) . From the amino a c i d composition, the 8 TpIII-b p e p t i d e corresponds t o the 27-30 r e s i d u e o f B ^ p I I I ( F i g . 27) w h i l e the B E T p I I I - a shows the amino a c i d a n a l y s i s corresponding to the 18-3L6 r e s i d u e 114 Origin P l a t e 26. T r y p t i c p e p t i d e maps o f AEB and AE$ w i t h n i n h y d r i n s t a i n . Normal 18 22 26 27 30 Val-Asn-Val-Asp-Glu-Val-Gly-Gly-Glu-Ala-Leu-Gly-Arg B^pIII . >. Mutant 18 22 26 27 Val-Asn-Val-Asp-Glu-Val-Gly-Gly-Lys-Ala-Leu-Gly-Arg -<? 6 E T p I I I - a >\<— B ETpIII-b-?- £ Ul F i g u r e 27. A diagram o f known sequence of B^TpIII and the amino a c i d E a l t e r a t i o n o f the slow 6 mutant r e s u l t i n g 8 T p I I I - a and 8 ETpIII-b obtained. 116 o f B ^ T p I I I e x c e p t f o r one l e s s r e s i d u e o f g l u t a m i c a c i d and an a d d i t i o n a l r e s i d u e o f l y s i n e (Table V I I ) . Thus t h e r e p l a c e -ment o f g l u t a m i c a c i d by l y s i n e was su g g e s t e d . As r e g a r d s t o the known sequence o f B ^ p I I I ( F i g . 27) t h e r e p l a c e m e n t might o c c u r a t g l u t a m i c a c i d r e s i d u e 22 o r 26. However, t h e amino a c i d a n a l y s e s i n d i c a t e d t h a t t h e l a t t e r was t h e s i t e o f m u t a t i o n . I n c o n c l u s i o n , b i o c h e m i c a l i n v e s t i g a t i o n o f t h e s l o w 8 mutant r e v e a l e d t h e g l u t a m i c a c i d o f r e s i d u e 26 o f th e B - c h a i n was a l t e r e d t o l y s i n e . T h i s m u t a t i o n was i d e n t i c a l t o t h a t p r e v i o u s l y d e s i g n a t e d HbE ( a ^ B i 6 L ^ S ) ( 1 4 9 , 1 5 0 ) w h i c h i s h i g h l y p r e v a l e n t i n S o u t h - E a s t A s i a e s p e c i a l l y i n T h a i l a n d (146,147,148). However, a mutant known as H b E S a s k a t o o n ( a A g 2 2 G l u - L y s } ( 1 5 1 ) found i n a S c o t t i s h f a m i l y (151) i s v e r y s i m i l a r t o HbE ( a A B i 6 L ^ S ) i n terms o f t h e m o b i l i t y on s t a r c h g e l e l e c t r o p h o r e s i s , t h e amino a c i d a l t e r a t i o n and m u t a t i o n l o c a t e d a t B T p I I I . S i n c e S a s k a t o o n th e m u t a t i o n , HbE might p o s s i b l y o c c u r among the South-E a s t A s i a p o p u l a t i o n , t h e c h a r a c t e r i z a t i o n o f HbE ( a A B i 6 L y S ) based s o l e l y on e l e c t r o p h o r e t i c m o b i l i t y and s t a t i s t i c e v i d e n c e c o u l d g i v e a s p u r i o u s d i a g n o s i s i n some ca s e s u n l e s s t h e b i o c h e m i c a l i n v e s t i g a t i o n was a l s o c a r r i e d o u t . 5. To l o c a t e t h e s i t e o f m u t a t i o n o f the f a s t B - c h a i n v a r i a n t NY Two m i l l i g r a m s o f AEB was p e p t i d e mapped by HVE pH 6.5 i n t h e f i r s t d i m e n s i o n and d e s c e n d i n g chromatograph (120) i n th e second and compared w i t h t h a t o f the normal AEB-chain. N i n h y d r i n f o l l o w e d by t h e P a u l y s t a i n were employed t o l o c a t e t h e p e p t i d e s . The chromatograms o f b o t h normal and t h e v a r i a n t 117 E E Table V I I . Amino a c i d analyses o f 3 T p I I I - a and 8 TpIII-b peptide E from the p e p t i d e map of AEB B E T p I I I - a umoles re s i d u e s 3 ETpIII-b umoles re s i d u e s Lys H i s Arg 0.096 0.92 (1) 0.006 0 .095 0.05 (0) 0.82 (1) Asp Thr Ser Glu Pro Gly A l a V a l Met Leu T y r 0 .229. 2.17 (2) 0.009 0.08 (0) 0 .102 0 .211 0.309 0.96 (1) 2.09 (2) 2.94 (3) 0 .125 0.136 0.116 1.07 (1) 1.17 (1) 1.00 (1) 118 are shown on P l a t e 28. By comparison w i t h the normal t r y p t i c p e p t i d e of the B-chain ( F i g . 12a, and P l a t e 28) i t can be seen t h a t the Pauly p o s i t i v e p e p t i d e ;BTpXII-b of the v a r i a n t i s d i s p l a c e d towards the anode. T h e r e f o r e , the amino a c i d a l t e r -a t i o n which produces a g r e a t e r net n e g a t i v e charge occurs between r e s i d u e s 113-120 of 8-chain. In o r d e r t o determine the amino a c i d s u b s t i t u t i o n , the A NY p e p t i d e s o b t a i n e d by mapping 5 mg o f t r y p t i c AEB and AEB were l o c a t e d by l i g h t n i n h y d r i n s t a i n i n g , e l u t e d w i t h 6N HCl and h y d r o l y z e d under vacuum a t 110° C f o r 24 hours. The h y d r o l y s a t e was s u b j e c t e d to amino a c i d a n a l y s i s (Table V I I I ) . The amino a c i d composition o f B ^ p X I I - b showed i t t o r e p r e s e n t NY r e s i d u e s 113-120 o f the B-chain w h i l e B TpXII-b showed no v a l i n e and an a d d i t i o n a l g l u t a m i c a c i d (Table V I I I ) . T h i s i n d i c a t e d t h a t v a l i n e a t r e s i d u e 113 was a l t e r e d t o g l u t a m i c NY a c i d or glutamme. The m o b i l i t y o f the a l t e r e d p e p t i d e B TpXII-b d u r i n g HVE pH 6.5 r e v e l e d a g r e a t e r net n e g a t i v e charge w h i l e NY $ g l o b i n c h a i n ( F i g . 25) r e q u i r e d l e s s s a l t f o r e l u t i o n from C M - c e l l u l o s e . T h i s i s s t r o n g l y s u g g e s t i v e of v a l i n e b e i n g r e p l a c e d by g l u t a m i c a c i d r a t h e r than glutamine, however, an unambiguous answer c o u l d be o b t a i n e d by enzymatic h y d r o l y s i s NY o f B TpXII-b i n t o f r e e ammo a c i d s f o l l o w e d chromatography on a s i n g l e column system (136,137). NY The B TpXII-b from t h r e e p e p t i d e maps of 5 mg amount of NY t r y p t i c AEB were l o c a t e d by l i g h t n i n h y d r i n s t a i n i n g and e l u t e d w i t h water ( I t would be noted t h a t the chromatogram had 119 P l a t e 28. T r y p t i c p e p t i d e maps of normal and v a r i a n t AE6 c h a i n w i t h n i n h y d r i n s t a i n . 120 A MY Table V I I I . Amino a c i d analyses of 8 TpXII-b and 6 TpXII-b which were e l u t e d from the pe p t i d e maps o f 5 mgm A NY o f t r y p t i c p e p t i d e AEB and AEB . B ^ p X I I - b B N Y T p X I I - b ymoles r e s i d u e s ymoles r e s i d u e s Lys His Arg 0.044 0.093 0.98 (1) 2.07 (2) 0 .054 0 .129 0.81 (1) 1.90 (2) Asp Thr Ser Glu 0 .006 0 .009 0 .061 0.08 (0) 0.13 (0) 0.87 (1) Pro Gly A l a V a l Met Leu Tyr Phe 0.049 0.050 0 .042 0.037 0.044 1.11 (1) 1.13 (1) 0.95 (1) 0.83 (1) 0.97 (1) 0 .068 0.074 0 0.070 0 .077 1.01 (1) 1.10 (1) 0 1.04 (1) 1.14 (1) 121 to be completely d r i e d and f r e e d from chromatographic s o l v e n t , otherwise the mixing o f o r g a n i c s o l v e n t w i t h water would e l u t e the n i n h y d r i n c o l o r o f f the paper and contaminate the p e p t i d e s o l u t i o n ) . The c l e a r p e p t i d e s o l u t i o n was l y o p h i l i z e d . One t h i r d o f the p e p t i d e , approximately 0.5 umoles, was h y d r o l y z e d w i t h pronase (Calbiochem) i n 0.1M NIUHCO3 a t 37° C f o r 48 hours at the enzyme s u b s t r a t e r a t i o about 1/20. The h y d r o l y s a t e was l y o p h i l i z e d and s u b j e c t e d to amino a c i d a n a l y s i s . Low y i e l d s o f f r e e amino a c i d s were ob t a i n e d a f t e r h y d r o l y s i s , n e v e r t h e l e s s , a peak c o r r e s p o n d i n g to g l u t a m i c a c i d d i d appear i n the p r o f i l e . The o t h e r one t h i r d o f the p e p t i d e was in c u b a t e d w i t h 5 mi c r o -l i t r e s o f l e u c i n e amino p e p t i d a s e (LAP)(9 mg/ml, Worthington, Biochem Corp) i n 0.2M ammonium a c e t a t e , pH 8.8 c o n t a i n i n g 0.005M MgCl2 a t 37° C f o r 18 hours. The h y d r o l y s a t e was d r i e d i n a r o t a r y e v a p o r a t o r and dry r e s i d u e was d i s s o l v e d i n water and d r i e d a g a i n . The h y d r o l y s a t e was a p p l i e d t o the amino a c i d a n a l y z e r . The r e s u l t s o f f r e e amino a c i d r e l e a s e from LAP (Table IX) not only confirmed g l u t a m i c a c i d as the replacement, but a l s o suggested the sequence o f p e p t i d e from the N-terminus from the i n d i v i d u a l y i e l d s o f f r e e amino a c i d s . Thus the glu t a m i c a c i d w i t h the h i g h e s t y i e l d appeared t o be the N-terminal amino a c i d o f the mutant p e p t i d e . T h i s evidence, t h e r e f o r e , s u b s t a n t i a t e s the s i t e o f mutation as o c c u r i n g a t p o s i t i o n 113, by the replacement o f v a l i n e f o r g l u t a m i c a c i d i n 8-chain (see F i g . 29). 122 Table IX. Amino a c i d a n a l y s i s o f f r e e amino a c i d l i b e r a t i o n from NY the h y d r o l y s i s o f 6 TpXII-b with LAP umoles of r e l e a s e number residue i n the peptide Lys 0.027 1 His 0.075 2 Arg Asp Thr Ser Glu 0.0 61 1 Gin Pro Gly 0 .038 1 Al a 0.055 1 V a l Met Leu 0.058 1 Tyr Phe 0.046 1 Normal A E g ^ p X I I (8 ATpXII-a + 8 ATpXII-b) 105 111 113 120 Leu-Leu-Gly-Asn-Val-Leu-Val-SAC-Val-Leu-Ala-His-His-Phe-Gly-Lys <sf A E g ^ p X I I 9* -< ; 8 ATpXII-a ^l^sr S ^ p X I I - b ^ Mutant 113 120 Glu-Leu-Ala-His-His-Phe-Gly-Lys i N Y I 8 TpXII-b F i g u r e 29. A diagram of 8 ATpXII-a and 8 ATpXII-b r e s u l t e d the t r y p s i n cleavage of the A E B ^ p X I I and the amino a c i d a l t e r a t i o n N Y o f 8 TpXII-b. SAC=S-aminoethylcystein 124 The N - t e r m i n a l amino a c i d s o f the g TpXII-b and the normal g ^ p X I I - b were determined by D a n s y l a t i o n (132) and the DNS-amino a c i d s i d e n t i f i e d on t h i n l a y e r chromatography as d e s c r i b e d by Black and Dixon ( 1 3 3 ) ( P l a t e 30). I t may be seen t h a t the DNS-valine i s the N-terminal amino a c i d o f the normal p e p t i d e as d e t e c t e d i n the a p o l a r s o l v e n t w h i l e the DNS-glutamic a c i d s e p a r a t e d i n the p o l a r s o l v e n t , was o b t a i n e d from the v a r i a n t p e p t i d e s . Summarizing the b i o c h e m i c a l s t u d i e s of the f a s t g-chain v a r i a n t , i t i s e v i d e n t t h a t the s i t e o f mutation i n which the v a l i n e of r e s i d u e 113 i s r e p l a c e d by glutamic a c i d i s i d e n t i c a l t o the p r e v i o u s l y named Hb New York ( a A g l 1 sVal-KSlUj ( g C j ) ^ Hb New York was f i r s t d e s c r i b e d by Ranney, Jacobs and Nagel (69) i n a Chinese-American f a m i l y i n heterozygotes and i n combination w i t h g-thalassemia. The l a t t e r appeared t o i n t e r a c t and m a n i f e s t the c l i n i c a l f i n d i n g s of h e m o l y t i c anemia with A almost complete d e p r e s s i o n of the g s y n t h e s i s . In t h i s study, i t was entercounted i n a T h a i f a m i l y , furthermore, a combination with HbE was a l s o observed i n two cases. C. The R e l a t i o n Of Amino A c i d A l t e r a t i o n s To Hemoglobin S t r u c t u r e From the X-ray c r y s t a l l o g r a p h i c s t u d i e s of hemoglobin, i t i s thought t h a t the glutamic a c i d a t p o s i t i o n 26 of g-chain, B8g ( r e s i d u e No. 8 h e l i c a l B segment of g-chain) hydrogen bonds with the h i s t i d i n e Gl8g and G19g. Furthermore i t a l s o approaches c l o s e t o a r g i n i n e Bl2g (49,77). As a l r e a d y shown ( F i g . 5), the 125 A p o l a r s o l v e n t system DNS-Val 1 2 3 4 5 6 7 8 9 10 11 P l a t e 30. T h i n l a y e r chromatography of DNS-amino a c i d s 1 = H i s , 2 = Asp; 3,7 = Glu; 4,8 = DNS-amino a c i d from 3~ TpXII-b; 5,9 = DNS-amino a c i d from 3 TpXII-b; 6,10 = V a l ; 11 = Leu. 126 a r g i n i n e B12$ and h i s t i d i n e G18 are i n v o l v e d i n hydrogen bonding of a1-^1 c o n t a c t . Thus, the replacement of glutamic a c i d by l y s i n e i n the case of HbE, i n t r o d u c e s a p o s i t i v e charge which would d i s t u r b the i n t e r a c t i o n and hence weaken the a 1 - ^ 1 bonding. A c c o r d i n g t o Perutz e t a l (48) , the v a l i n e o f r e s i d u e 113 of the 3-chain, G158 and the n e i g h b o r i n g a l a n i n e G173 occur a t the s u r f a c e c r e v i c e which i s a b a s i c r e g i o n because o f a r g i n i n e B123, h i s t i d i n e G18g and h i s t i d i n e G198. The r e s i d u e s : G178, G183 and B128 are known t o form hydrogen bonds i n a 1 - ^ 1 s u b - u n i t c o n t a c t . Hence, i n the glutamic a c i d s u b s t i t u t i o n f o r v a l i n e G158 i n Hb New York, the c a r b o x y l group might cause the u n s t a b i l i t y of t h i s s u b - u n i t i n t e r a c t i o n , i n a s i m i l a r way t h a t was found i n HbE (69,77). I t i s i n t e r e s t i n g t o note t h a t the m o b i l i t y o f Hb New York on a s t a r c h g e l e l e c t r o p h o r e s i s pH 6.8 ( P l a t e 23) was s l i g h t l y g r e a t e r than t h a t o f Hb A i n s p i t e o f p o s s e s s i n g an a d d i t i o n a l n e g a t i v e charge from the a l t e r a t i o n o f v a l i n e to glutamic a c i d . NY However, the m i g r a t i o n r a t e of f r e e 3 cha i n on hemoglobin d i s s o c i a t i o n with PHMB ( P l a t e 24) arid i t s e l u t i o n b e h a v i o r on C M - c e l l u l o s e chromatography ( F i g . 25) are i n agreement w i t h the charge change. These f i n d i n g s suggest t h a t the c a r b o x y l group of glutamic a c i d might i n some way i n t e r a c t with the b a s i c r e s i d u e s , e s p e c i a l l y the f r e e i m i d a z o l e o f h i s t i d i n e G198 i n the deep s u r f a c e o f the c r e v i c e (69,77) r e s u l t i n g i n a decreased net charge of the t e t r a m e r i c hemoglobin molecule. In c o n t r a s t , the f r e e c a r b o x y l group o f glutamic a c i d i n the monomer of the 127 mutant was f u l l y i o n i z e d l e a d i n g t o a d i s t i n c t change i n the m o b i l i t y on s t a r c h g e l e l e c t r o p h o r e s i s i n hemoglobin d i s s o c i a t i o n study w i t h PHMB and i n the e l u t i o n p r o f i l e from C M - c e l l u l o s e chromatography. I t was e v i d e n t t h a t the s e q u e n t i a l d i s s o c i a t i o n of the hemoglobin molecule from a tetramer t o dimers and f i n a l l y t o monomers i n v o l v e s d i s r u p t i o n s o f the a 1 - 8 2 and c t 1 ^ 1 s u b - u n i t c o n t a c t r e s p e c t i v e l y (56). Thus i n HbE and Hb New York which are thought to be due t o a derangement o f o^-B 1 c o n t a c t one would expect t o f i n d an i n c r e a s e d d i s s o c i a t i o n o f dimers i n t o monomers. However, t h i s type o f d i s s o c i a t i o n does not occur at p h y s i o l o g i c a l pH and s a l t c o n c e n t r a t i o n although i t can be seen under extreme c o n d i t i o n s (62). T h e r e f o r e , a heterozygote of HbE and Hb New York as w e l l as a double heterozygote o f both v a r i a n t s s h o u l d not show any remarkable c l i n i c a l or he m a t o l o g i c a l a b n o r m a l i t i e s , a p r e d i c t i o n which i s c o n s i s t e n t w i t h the pr e s e n t s t u d i e s . D. D i s c u s s i o n In T h a i l a n d there e x i s t s a h i g h i n c i d e n c e of thalassemias and abnormal hemoglobins(104,146,147,148). At the pr e s e n t time, b e s i d e s HbH (B A) and Hb Ba r t ' s (y^) r e s u l t i n g from a-thalassemias a number o f o t h e r mutants have been d e s c r i b e d namely: H b E ( a A 3 i 6 L y (149,150), H b J B a n ^ k o k ( a A 6 l 6 A s P ) ( 7 3 , 7 4 ) , HbD P u n=> a b ( a A B l 2 l G l n ) (152), Hb S i r i r a j ( a A 8 l L y s ) ( 1 5 3 ) , HbQ (not y e t c h a r a c t e r i z e d ) and High F gene (20) . In the presen t s t u d i e s , two more abnormal 128 hemoglobins; Hb New York (0 :282 1 3 ) and Hb Mahidol ( a l " 8 2 ) are d e s c r i b e d . Other mutants d e t e c t e d d u r i n g the s t u d i e s o f hemoglobinopathies and r e l a t e d problems a t the D i v i s i o n of Hematology, Department o f Medicine, S i r i r a j M e d i c a l S c h o o l , Bangkok are w a i t i n g b i o c h e m i c a l c h a r a c t e r i z a t i o n . S i n c e there i s a remarkably h i g h i n c i d e n c e o f HbE ( a A 8 l 6 L y S ) i n the T h a i p o p u l a t i o n , the n a t i o n a l average i s 13% but i n some areas i t may reach 50% (104), i t i s not s u r p r i s i n g t h a t HbE i s found i n combination w i t h a number of other genes, producing a spectrum of c l i n i c a l syndromes some o f which have been d e s c r i b e d prev-i o u s l y (103,104,107) and are summarized i n T a b l e I . The present s t u d i e s d e s c r i b e d a new combination of HbE and Hb New York i n a T h a i f a m i l y i n c l u d i n g g e n e t i c a l , h e m a t o l o g i c a l and b i o c h e m i c a l s t u d i e s . Although t h e r e i s i n d i r e c t evidence to suggest t h a t both v a r i a n t s have deranged cx^-B1 s u b - u n i t i n t e r -a c t i o n s , the double heterozygotes appeared h e a l t h y , a t l e a s t f o r the two cases examined. E. Summary A combination of a slow hemoglobin - b e l i e v e d t o be HbE and a f a s t hemoglobin was d e t e c t e d on a s t a r c h g e l e l e c t r o p h o r e s i s of a hemolysate d u r i n g the r o u t i n e hemoglobin type examination of a new p a t i e n t a t D i v i s i o n o f Hematology, Department of Medicine, S i r i r a j H o s p i t a l , Bangkok. The p r o p o s i t u s , a 22 yea r o l d T h a i male, showed no c l i n i c a l o r h e m a t o l o g i c a l a b n o r m a l i t i e s except f o r the presence o f the abnormal pigments. The f a m i l y 129 study showed the segregation of HbE and the fa s t hemoglobin variant i n the other members. Like the propositus, one of his brothers was also found to be doubly heterozygous for both variants. The biochemical characterization of both variants from the hemolysate of the propositus was carr i e d out. The hemoglobin chain d i s s o c i a t i o n studies suggested that the double heterozygote gave r i s e to a slow $ mutant and a fa s t 3 variant. The separation of both 8 mutant chain was achieved by CM-cellulose chromatography. The s t r u c t u r a l characterization of the is o l a t e d 8 slow variant showed i t to be i d e n t i c a l to the previously reported HbE (ct A8 2 e L y s)(149,150) while the fast 8 variant was found to be the same as Hb New York ( a A 8 2 1 3 G 1 U ) ( 6 9 ) . Both abnormal pigments are thought to a f f e c t i n d i r e c t l y the contact of the o^-B1 sub-units r e s u l t i n g i n an increased d i s s o c i a t i o n of dimers to monomer sub-units. 130 V I I . HEMOGLOBIN MAHIDOL - A NEW a-CHAIN MUTANT During s t u d i e s o f hemoglobinopathies and r e l a t e d problems i n T h a i l a n d by the D i v i s i o n o f Hematology, Department of Medicine, S i r i r a j M e d i c a l S c h o o l , Mahidol U n i v e r s i t y , Bangkok, a number of r a r e abnormal hemoglobins were d e t e c t e d among T h a i and Chinese p o p u l a t i o n on s t a r c h g e l e l e c t r o p h o r e s i s w i t h t r i s - E D T A -b o r a t e b u f f e r pH 8.6. Some have a l r e a d y been c h a r a c t e r i z e d e.g. H b J B a n g k o k ( a A 6 l 6 A s p ) (73,74), H b D P u n j a b (a A 3 i 2 1 G l n ) (152) , Hb New York ( a A 3 l 1 3 G ^ U ) ( s e e the p r e v i o u s s e c t i o n ) . The presen t s t u d i e s d e s c r i b e a slow hemoglobin obtained from t h r e e u n r e l a t e d p a t i e n t s ( S . J . , S.T. and S.R.) i n Bangkok. The r e s u l t s o f s t r u c t u r a l s t u d i e s on a l l three r e v e a l e d an i d e n t i c a l amino a c i d a l t e r a t i o n i n which r e s i d u e 74 o f cx-chain i s changed from an a s p a r t y l t o a h i s t i d y l r e s i d u e . T h i s mutation has not been p r e v i o u s l y d e s c r i b e d . I t i s proposed t h e r e f o r e t o name t h i s new hemoglobin ( a 2 I * H l S 0 A ) Hb Mahidol, a f t e r Mahidol U n i v e r s i t y i n Bangkok. A. C l i n i c a l , H e m a t o l o g i c a l and G e n e t i c a l Study  S.J. Case S.J., the p r o p o s i t u s , a 42 year o l d Chinese man was admitted t o S i r i r a j H o s p i t a l complaining o f edema and shortage of b r e a t h . He had s u f f e r e d from c h r o n i c anemia and jaundic e s i n c e c h i l d h o o d . A p h y s i c a l examination r e v e a l e d a hyposthenic appearance, marked anemia, m i l d j a u n d i c e , and 131 hepatosplenomegaly. The h e m a t o l o g i c a l f i n d i n g s on admission (9/5/67) d i s c l o s e d marked hemolysis and severe t h a l a s s e m i c a b n o r m a l i t i e s o f the red b l o o d c e l l s (Table X ) . In methylene blu e p r e p a r a t i o n 85% of the red c e l l s c o n t a i n e d i n c l u s i o n b o d i e s . Furthermore, the e l e c t r o p h o r e s i s showed a f a s t hemoglobin -b e l i e v e d t o be HbH, a slow hemoglob i n and no HbA. The i n i t i a l d i a g n o s i s o f hemoglobinopathies - a-thalassemia and a mutant with c o m p l i c a t i o n s due to h e m o l y t i c c r i s i s and c o n g e s t i v e h e a r t f a i l u r e was made. A f t e r the c r i s i s d i sappeared the hemoglobin l e v e l was maintained around 10 gm% (see T a b l e X) w i t h a moderate degree o f hemolysis. Four members o f h i s f a m i l y were examined, the pedigree and h e m a t o l o g i c a l f i n d i n g s are shown on F i g u r e 31 and T a b l e X. His mother 1-2 and son I I I - 2 were found t o e x h i b i t the a-thalassemia w h i l e I I I - l was a heterozygote o f Hb Mahidol showing no c l i n i c a l o r h e m a t o l o g i c a l a b n o r m a l i t i e s besides the presence o f the abnormal pigment on s t a r c h g e l a n a l y s i s . S.T. and S.R. Cases S.T. - a 42 year o l d T h a i - C a u c a s i a n woman and S.R. - a T h a i female aged 38 y e a r s from u n r e l a t e d f a m i l i e s were found t o be heterozygous f o r Hb Mahidol. Both S.T. and S.R. appeared h e a l t h y and were h e m a t o l o g i c a l l y normal except f o r the presence of 22.9% and- 25.3% of abnormal pigment r e s p e c t i v e l y . B. B i o c h e m i c a l I n v e s t i g a t i o n The packed r e d c e l l s o f S.J., S.T., and S.R. were washed wit h 0.9% NaCl, f r o z e n i n d r y - i c e and t r a n s p o r t e d t o Vancouver. The hemolysates were prepared as p r e v i o u s l y d e s c r i b e d . T a b l e X. Summary o f h e m a t o l o g i c a l f i n d i n g s o f S.R. f a m i l y . Pedigree Age Hb RBC PCV I n c l u - RBC s i o n Morphology Body Serum Hb type % % % Abnormal Iro n HbA 2 A l k a l i Hb Denat. D e s i g n a t i o n £-2 60 13.3 6.69 42 Hypochro. + micro. + A + A 2 2.2 0.38 otThal. t r a i t EI-1 35 10.0 4 .36 31 apparently normal 21.4 A + A 2 2.3 0.28 ' Fe d e f i c i e n c y 42 9/5/67 2.8 1.08 10 85% Severe degree 160 of Thalassemic abnormali t i e s H+Mahidol 0 .80 H = 11.6% CI-2 26/6/67 9.3 4.57 35 otThal-Hb Mahidol 26/8/67 10.8 5.36 37.5 99% Moderate degree of Thalassemic changes H+Mahidol 0 .50 H = 21.45% 19/1/70 10.1 5.21 38.5 LII-1 16 14.6 5.01 47 0 Normal A+Mahidol 2.57 0 +A+9low A 2 .48 Mahidol=25.2% Hb Mahidol t r a i t slow A 2=1.3% [II-2 13 12.3 5.74 40 0 Hypochromia + 78.5 Mi c r o c y t e + A+A2 2.2 0 .40 otThal t r a i t CO 133 n i I i D l L IL_ 0 \ i \ * 0 ( 1 oc thalassemia trait ot thalassemia Mb Mahidol [ D _] Dead 1 CD ( > Not tested Hb Mahidol trail Normal F i g u r e 31. Pedigree of a-Thaiassemia Hb Mahidol o f S.J. (II-2) f a m i l y . 134 1. Hemoglobin e l e c t r o p h o r e s i s and hemoglobin c h a i n d i s s o c i a t i o n s t u d i e s Hemolysates from t h r e e p a t i e n t s ( S . J . , S.T. and S.R.) were s u b j e c t e d t o s t a r c h g e l e l e c t r o p h o r e s i s ( P l a t e 32). S.J. showed only two major bands: a slow mutant hemoglobin d e s i g n a t e d as Hb Mahidol and a f a s t hemoglobin - b e l i e v e d t o be HbH (3i») , t o g e t h e r w i t h a very s m a l l amount of a slow hemoglobin A 2 . Normal HbA and HbA2 were not d e t e c t a b l e by b e n z i d i n e s t a i n i n the S.J. hemolysate. Both S.T. and S.R. showed two bands of hemoglobin which had the same m o b i l i t y as those i n the S.J. hemolysate namely, a slow hemoglobin -Hb Mahidol and a slow HbA2, i n a d d i t i o n t o the normal HbA and HbA2 ( P l a t e 32). There are a t l e a s t two p i e c e s o f evidence s u g g e s t i n g t h a t Hb Mahidol i s a slow a-chain mutant. F i r s t l y , i t i s a s s o c i a t e d w i t h a slow HbA 2 which i s l i k e l y t o r e p r e s e n t Mahidol r A 2 0 ,. .. . , . . ct2 02 . Secondly, i t i n t e r a c t s , i n the case of S.J., with the a-thalassemia gene whose presence i s i n d i c a t e d by the o b s e r v a t i o n of HbH (£„) d u r i n g s t a r c h g e l e l e c t r o p h o r e s i s and the d e t e c t i o n i n red c e l l s of i n c l u s i o n b o d i e d (154) and t h a l a s s e m i c changes. T h i s i n t e r a c t i o n l e a d s t o complete d e p r e s s i o n of normal a-chain s y n t h e s i s and r e s u l t s i n a complete absence of HbA on s t a r c h g e l e l e c t r o p h o r e s i s ( P l a t e 32). To c o n f i r m Hb Mahidol as a slow a-chain v a r i a n t , the hemoglobin chains were d i s s o c i a t e d w i t h PHMB (P l a t e 33). 135 Slow HbA2 HbA2 HbA t 4 i Anode O r i g i n Hb Mahidol HbH P l a t e 32. Hemoglobin s t a r c h g e l e l e c t r o p h o r e s i s , t r i s - E D T A -b o r a t e b u f f e r , pH 8.6, w i t h b e n z i d i n e s t a i n . 136 Plate 3 3 . Hemoglobin chain d i s s o c i a t i o n with PHMB treatment on starch gel electrophoresis, tris-EDTA-borate buffer, pH 8 . 6 , i n the presence of PHMB. 137 I t i s i n t e r e s t i n g to note t h a t S.J. r e v e a l s a g-chain w i t h normal e l e c t r o p h o r e t i c m o b i l i t y and a t o t a l absence of the ct A-chain which i s r e p l a c e d by the slow a M a n i d ° l v a r i a n t . Both S.T. and S.R. showed a slow a-chain w i t h a m o b i l i t y i d e n t i c a l t o a M a n ^ d o 1 t o g e t h e r with some normal g A and a A - c h a i n . 2. S t r u c t u r a l c h a r a c t e r i z a t i o n of Hb Mahidol i n S.J. sample G l o b i n , prepared from the S.J. hemolysate, was f r a c -t i o n a t e d by C M - c e l l u l o s e chromatography i n 8 M urea, 0.05 M g-mercaptoethanol and sodium phosphate b u f f e r (74) . As seen i n F i g . 34, the e l u t i o n p r o f i l e showed only two major c h a i n s : 8 A and a M a h l d o l / a r e s u l t c o n s i s t e n t with the PHMB c h a i n d i s s o c i a t i o n study. I t was a l s o noted t h a t the a M a n i d ° l e l u t e d a t a h i g h e r i o n i c s t r e n g t h than a , i n d i c a t i n g t h a t i t i s l e s s a n i o n i c . When t r y p t i c p e p t i d e s from both normal and mutant a-chain i n e i t h e r the AE or u n s u b s t i t u t e d form were compared by p e p t i d e mapping ( P l a t e 35) u s i n g both n i n h y d r i n and s p e c i a l s t a i n s , aTpIX, as w e l l as aTpVIII-IX, peptides c o n t a i n i n g both methionine ( C h l o r o p l a t i n a t e p o s i t i v e ) and h i s t i d i n e (Pauly p o s i t i v e ) , were seen t o migrate more r a p i d l y to the cathode i n the mutant than the normal s u g g e s t i n g t h a t the s i t e of mutation was i n aTpIX ( p o s i t i o n s 62-90 of the a - c h a i n ) . The a ^ p I X (aTpIX o f mutant) ran s l i g h t l y f a s t e r and a ^ p I X s l i g h t l y slower than e-DNP-Lysine on HV e l e c t r o p h o r e s i s a t pH 6.5 so t h a t t h i s marker c o u l d be used t o f o l l o w the progress o f an extended p r e p a r a t i v e HV e l e c t r o p h o r e s i s of these p e p t i d e s 138 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 | 6 F R A C T I O N N U M B E R F i g u r e 34. Chromatography of S.J. g l o b i n (210 mg) on C M - c e l l u l o s e i n 8M urea, 0.05M 6-mercaptoethanol and sodium phosphate b u f f e r . 139 * <»MT P VIII-IX O r i g i n P l a t e 35. T r y p t i c p e p t i d e maps o f non AEa c h a i n A = n o r m a l , B = v a r i a n t o f S.J. sample. 140 at pH 6.5 f o r 3 hours a t 3 kv. The p e p t i d e s were l o c a t e d by s t a i n i n g guide s t r i p s w i t h C h l o r o p l a t i n a t e and Pauly s t a i n s and the corresponding u n s t a i n e d paper s t r i p s were sewn i n t o a f r e s h sheet and r e - r u n a t pH 3.6. a^TpIX and a^TpIX were then e l u t e d w i t h d i l u t e NH^OH s o l u t i o n and h y d r o l y z e d . T h e i r amino a c i d analyses are shown i n T a b l e XI. I t can be seen t h a t t h e r e i s one l e s s a s p a r t i c a c i d (or asparagine) i n a ^ p I X and one more h i s t i d i n e thus s u g g e s t i n g an Asp (Asn)-*His p o i n t mutation. aTpIX i s o l a t e d from the normal and the v a r i a n t was r e a c t e d w i t h cyanogen bromide (74,124) and s u b j e c t e d t o h i g h v o l t a g e e l e c t r o p h o r e s i s at pH 6.5. Three n i n h y d r i n p o s i t i v e , Pauly p o s i t i v e and methionine n e g a t i v e spots r e s u l t e d ( P l a t e 36). As expected, methionine at r e s i d u e 76 i n a^TpIX was c l e a v e d and two p e p t i d e s were o b t a i n e d : CNBr II-A, an a c i d i c p e p t i d e c o n t a i n i n g r e s i d u e s 62-76; Val- A l a - A s p - A l a - L e u - T h r - A s n - A l a - V a l -A l a - H i s - V a l - A s p - A s p - H s l * and CNBr I I I - A a b a s i c p e p t i d e com-p r i s i n g r e s i d u e s 77-90; Pro-Asn-Ala-Leu-Ser-Ala-Leu-Ser-Asp-L e u - H i s - A l a - H i s - L y s ( F i g . 37). CNBr I-A and CNBr I-M showed the same m o b i l i t y i n r e l a t i o n t o e-DNP-Lysine as the s t a r t i n g p e p t i d e s and probably r e p r e s e n t methionine s u l f o x i d e d e r i v a t i v e s of the s t a r t i n g p e p t i d e s which would be r e s i s t a n t t o cyanogen bromide cleavage. In the case o f a ^ p I X ; the b a s i c p e p t i d e , CNBr III-M, was i d e n t i c a l i n m o b i l i t y w i t h CNBr-III-A s u g g e s t i n g t h a t the r e s i d u e s between p o s i t i o n s 77-90 were unchanged. * H s l = Homoserine l a c t o n e 141 Table XI. The amino a c i d a n a l y s i s of aTpIX o f normal and v a r i a n t a ATpIX r A p I X umoles Residues umoles Residues Lys 0.049 1.16(1) 0.081 1.08(1) His 0.113 2.69(3) 0.277 3.7 (4) Arg Asp 0.260 6.19(6) 0.382 5.1 (5) Met S0 2 Trace Trace Thr 0.043 1.02(1) 0 .070 0.93(1) Ser 0.091 2.16(2) 0.140 1.86(2) Glu Pro 0.030 0.71(1) 0 .073 0.97(1) H a l f c y s t Gly A l a 0.277 6.6 (7) 0 .502 6.7 (7) V a l 0.137 3.2 (3) 0.231 3.08(3) Met Trace (1) 0.035 0.46(1) H e Leu 0.182 4.3 (4) 0.276 3.68(4) T y r Phe 142 Plate 36. Paper electrophoresis pH 6.5 of Mcyanogen bromide cleaved peptide of a TpIX and a TpIX. Normal -* a ATpIX • . > 62 64 68 74 75 76 77 90 Val-Ala-Asp-Ala^eu-Thr-Asn-Ma^al-A]a-H CNBr II-A ^ (-* CNBr I I I - A CNBr I-A ( o x i d i z e d form) Mutant f r A p I X . — — > 52 64 68 74 75 76 77 90 Val-Ala-Asp^a-Leu^hr-Asn-Ala-^al-Ala-His-^al-His-Asp-Met-Pro-Asn-Ala-Leu-Ser-Ala-Leu-Ser-Asp-Leu-His-Ala-H CNBr II-M CNBr III-M <<= CNBr I-M ( o x i d i z e d form) F i g u r e 37. A diagram o f cyanogen bromide cleavage of ct^FpIX and ct^TpIX. The amino a c i d sequence of a TpIX i s based on the study of H i l l and Konigsberg (37). 144 However, CNBr II-M o f the v a r i a n t , i n s t e a d o f b e i n g a c i d i c , now moved t o the n e u t r a l r e g i o n near the marker e-DNP-Lysine. Thus, the s i t e of a l t e r a t i o n must be i n t h i s p e p t i d e and an a s p a r t i c a c i d or asparagine between p o s i t i o n s 62-76 of a ^ p I X must have been s u b s t i t u t e d by h i s t i d i n e . Repeated attempts t o i s o l a t e s u f f i c i e n t amounts o f a^TpIX and a ^ p I X were made but only low y i e l d s were o b t a i n e d probably because o f the l a r g e s i z e o f these peptides and t h e i r p r o p e n s i t y t o adsorb i r r e v e r s i b l y both t o columns and paper. However, cleavage w i t h both t r y p s i n and cyanogen bromide l e d t o the i s o l a t i o n i n good y i e l d o f the s m a l l e r fragments CNBr II - A and CNBr II-M. A M S o l u b l e t r y p t i c p e p t i d e s of non AEot and non AEa , a f t e r l y o p h i l i z a t i o n , were r e a c t e d d i r e c t l y w i t h cyanogen bromide (74,124) and the trypsin-cyanogen bromide p e p t i d e maps of A M A a and a , are shown on P l a t e 38. I t i s c l e a r t h a t the a c o n t r o l , r^TpIX (as w e l l as o^TpVTII-IX) seen i n t r y p t i c p e p t i d e maps ( P l a t e 35) had disappeared but new s p o t s , e s p e c i a l l y one co r r e s p o n d i n g t o CNBr II-A, an a c i d i c p e p t i d e , were p r e s e n t i n the trypsin-CNBr p e p t i d e maps ( P l a t e 38). In the case of the mutant, the co r r e s p o n d i n g CNBr II-M, i n s t e a d o f b e i n g a c i d i c , now moved towards the cathode i n the n e u t r a l p e p t i d e area as p r e v i o u s l y shown a f t e r cyanogen bromide cleavage o f M a TpIX ( P l a t e 36) . 145 P l a t e 38. P e p t i d e maps o f t r y p s i n and cyanogen bromide c l e a v a g e of..a c h a i n . A = non AEa (normal) , B = non AEa (mutant). 146 CNBr II-A and CNBr I I - M from a trypsin-CNBr p e p t i d e map A M o f 5 mg o f a and a., a f t e r l i g h t n i n h y d r i n s t a i n i n g , were cut out, e l u t e d w i t h 6N HCl and h y d r o l y z e d under vacuum a t 110°C f o r 24 hours. Amino a c i d a n a l y s i s o f CNBr II-A confirmed i t t o be the a c i d i c p e p t i d e c o n t a i n i n g r e s i d u e s 62- 76 of r/TpIX (Table X I I ) . The amino a c i d a n a l y s i s o f CNBr I I - M a l s o i n d i c a t e d t h a t an a s p a r t i c a c i d o r asparagine between p o s i t i o n s 62-76 of a a ^ p I X had been s u b s t i t u t e d by a h i s t i d i n e . CNBr II-A and CNBr I I - M were prepared on a l a r g e r s c a l e A M from trypsin-CNBr t r e a t e d peptides o f non AEa and non AEa u s i n g paper e l e c t r o p h o r e s i s a t pH 6.5 f o l l o w e d by descending paper chromatography (120). The l o c a t i o n o f the peptides were based on the guide s t r i p s s t a i n e d with n i n h y d r i n and P a u l y . The c o r r e s p o n d i n g u n s t a i n e d p e p t i d e s were d i r e c t l y e l u t e d w i t h 0.25 M a c e t i c a c i d . These e l u a t e s were then h y d r o l y z e d under vacuum a t 110°C f o r 24 hours i n the 0.25 M a c e t i c a c i d and the h y d r o l y s a t e s d r i e d under vacuum. The d i l u t e a c e t i c a c i d cleavage products from CNBr I I - A and CNBr I I - M were f r a c t i o n a t e d by paper e l e c t r o p h o r e s i s a t pH 1.9. I t can be seen ( P l a t e 39) i n the case of CNBr II-A, t h a t t h e r e were a t l e a s t 5 cadmium-ninhydrin p o s i t i v e s p o t s , namely A - l , A-2, A-3, A-4 and A-5. The corres p o n d i n g u n s t a i n e d p e p t i d e s were e l u t e d w i t h d i l u t e N H i j O H s o l u t i o n , l y o p h i l i z e d and then h y d r o l y z e d under vacuum w i t h c o n s t a n t b o i l i n g 5.7 N HCl a t 110°C f o r 24 hours. Amino a c i d a n a l y s i s o f A-2 and A-3 pe p t i d e s showed them t o be impure so both p e p t i d e s were 147 Tabl e X I I . The amino a c i d a n a l y s i s of the CNBr II-A and CNBr II-M which were e l u t e d w i t h 6N HCl a c i d from 5 mg of t r y p s i n -CNBr t r e a t e d p e p t i d e mapping a f t e r l i g h t n i n h y d r i n s t a i n , CNBr II-A CNBr II-M ymoles Residues ymoles Residues Lys His H s l * Arg Asp Thr Ser Hsr** Glu Pro H a l f c y s t Gly A l a V a l Met H e Leu T y r Phe 0.033 Present 0.130 0.036 0.015 0.141 0.094 0 .030 0.99(1) 3.96(4) 1.08(1) 4.29(4) 2.86(3T 0 .90(1) 0.142 Present 0.285 0.095 0.035 0.386 0.258 0 .103 1.6 (2) 2.8 (3) 1.03(1) 4.2 (4) 2.8 (3) 1.1 (1) Homoserine Lactone Homoserine CNBr II-M Amino acid markers CNBr II-A Origin co Gentian V i o l e t Slow portion of methylgreen P l a t e 39. The cadmium n i n h y d r i n s t a i n e d paper e l e c t r o p h o r e s i s pH 1.9 o f gui d e s t r i p s o f d i l u t e a c e t i c a c i d h y d r o l y s a t e o f C N B r l l - A and C N B r l l - M . 149 r e f r a c t i o n a t e d by paper e l e c t r o p h o r e s i s a t pH 3.6 u n t i l a g e n t i a n v i o l e t marker had moved 16 cm toward the cathode from the o r i g i n . A f t e r cadmium-ninhydrin s t a i n i n g of the guide s t r i p s , A-2 and A-3 p eptides were found t o c o n s i s t o f A-2-a, A-2-b and A-3-a, A-3-b, m i g r a t i n g r e s p e c t i v e l y , 4, 13, 9.5 and 11 cm from the o r i g i n . Based on amino a c i d a n a l y s e s , shown i n T a b l e X I I I , these peptides corresponded t o the f o l l o w i n g sequences; A - l to f r e e a s p a r t i c a c i d ; A-2-a t o p o s i t i o n s 62-64, Va l - A l a - A s p ; A-2-b t o p o s i t i o n s 65-67, Ala-Leu-Thr; A-3-a t o homoserine (from p o s i t i o n 76) which was d e t e c t a b l e both as homoserine and homoserine l a c t o n e on the amino a c i d a n a l y z e r u s i n g the s i n g l e column system (19, 20); A-3-b t o p o s i t i o n s 65-73, Ala-Leu-Thr-A s n - A l a - V a l - A l a - H i s - V a l ; A-4 t o p o s i t i o n s 62-63, V a l - A l a and A-5 to p o s i t i o n s 69-73, A l a - V a l - A l a - H i s - V a l r e s p e c t i v e l y ( F i g . 40). C o n s i d e r i n g the key p e p t i d e s A-4, A - l , A-2-b, A-5 and A-3-a as w e l l as A-2-a and A-3-b ( F i g . 40) o b t a i n e d from the p a r t i a l d i l u t e a c e t i c a c i d cleavage of CNBr II-A, h y d r o l y s i s o c c u r r e d , as expected, at a s p a r t y l r e s i d u e s 64, 74 and 75 as w e l l as a s p a r a g i n y l 68. As a l r e a d y mentioned, i t appeared from the composition of CNBr II-M t h a t an a s p a r t y l (or aspara-g i n y l ) had been changed t o a h i s t i d y l r e s i d u e i m p l y i n g t h a t the p o i n t of mutation might be a t an a s p a r t y l r e s i d u e a t p o s i t i o n s 64, 74, 75 o r an a s p a r a g i n y l a t r e s i d u e 68. In the case of the mutant the d i l u t e a c e t i c a c i d h y d r o l y s a t e o f CNBr II-M a f t e r HV e l e c t r o p h o r e s i s a t pH 1.9 showed 8 spots Table X I I I . Amino A c i d A n a l y s i s o f the D i l u t e A c e t i c A c i d Cleavage of CNBr II-A Pe p t i d e A - l A-2 A-3 A-4 A-5 0.074 0.83(1) 0.015 0.9 (1) A-2-a A-2-b A-3-a A-3-b umol. Residues umol. Residues umol. Residues umol. Residues umol. Residues umol. Residues umol. Residues Lys His H s l * P r e s e n t Arg Asp 0.150 rhr 3er flsr** 0.040 Glu 0.029 0.88(1) 0.026 0.89(1) 0.094 1.1 (1) 0.095 1.1 (1) Pro 31y Ma / a l det [ l e u Jeu Tyr >he 0.038 1.15(1) 0.035 1.20(1) 0.031 0.94(1) 0.025 0.86(1) 0.289 3.2 (3) 0.072 0.98(1) 0.036 2.1 (2) 0.167 1.87(2) 0.074 1.01(1) 0.033 1.9 (2) 0.092 1.0 (1) o Normal — CNBr II-A 5 * 62 64 65 67 68 69 74 75 76 V a l - A l a - Asp - Ala-Leu-Thr - Asn - A l a - V a l - A l a - H i s - V a l - Asp - Asp-Hsl A-2-a > k — A-2-b |*-A-l-*|-<= A-5 > |*A-l-^A-l>|*A-3-a* -s-A-4—A-l-?-|< A-3-b >\ Mutant -* CNBr II-M 62 64 65 67 68 69 74 75 76 V a l - A l a - Asp - Ala-Leu-Thr - Asn - A l a - V a l - A l a - H i s - V a l - H i s - Asp - H s l «M-4-a-*-|<-M-l->|-< M-2 3- |*M-l->|-< M-8 ^j-«-M-l^(<-M-3> U M-5 >f«M-l-*| H? M-4-b >t F i g u r e 40. Diagram of d i l u t e a c e t i c a c i d cleavage fragments of CNBr II-A and CNBr II-M cleavage. 152 s t a i n i n g w i t h cadmium n i n h y d r i n , d e s i g n a t e d as M-1, M-2, M-3 M-4, M-5, M-6, M-7 and M-8 ( P l a t e 39). Amino a c i d a n a l y s i s o f M-4 i n d i c a t e d inhomogeneity and the m a t e r i a l was r e f r a c -t i o n a t e d by paper e l e c t r o p h o r e s i s a t pH 3.6 u n t i l the g e n t i a n v i o l e t marker had migrated 23 cm from the o r i g i n . Cadmium n i n h y d r i n s t a i n i n g of guide s t r i p s showed two p e p t i d e s : M-4-a and M-4-b m i g r a t i n g a t 19 and 22.5 cm from the o r i g i n r e s p e c t i v e l y . S t u d i e s o f the N - t erminal amino a c i d s by d a n s y l a t i o n (132) and i d e n t i f i c a t i o n of DNS-amino a c i d s of u n f r a c t i o n a t e d M-4 on t h i n l a y e r chromatography (133), showed two N - t e r m i n a l s , DNS-Val and DNS-Ala; a f t e r r e f r a c t i o n a t i o n , M-4-a showed DNS-Val and M-4-b, DNS-Ala r e s p e c t i v e l y ( P l a t e 41). The amino a c i d a n a l y ses of M-1, M-2, M-3, M-4-a, M-4-b, M-5 and M-8 are shown on T a b l e XIV. A n a l y s i s of M-6 and M-7 i n d i c a t e d t h a t they were contaminants. From the amino a c i d compositions, M-4-a was the d i p e p t i d e , V a l - A l a (pos. 62-63), M-1 was f r e e a s p a r t i c a c i d , M-2, Ala-Leu-Thr (pos. 65-67) and M-3 was f r e e homoserine from p o s i t i o n 76. T h e i r r e l a t i o n s h i p s are shown i n F i g . 40. S ince the p e p t i d e s M-4-a, M-1, M-2 and M-3 from the mutant corresponded t o A-4, A - l , A-2-b and A-3-a r e s p e c t i v e l y , i t was u n l i k e l y t h a t the amino a c i d a l t e r a t i o n was l o c a t e d a t e i t h e r o f the a s p a r t y l r e s i d u e s a t p o s i t i o n s 64 o r 75 or the a s p a r a g i n y l at p o s i t i o n 68. However, the a s p a r t y l r e s i d u e a t p o s i t i o n 74 c o u l d be the s i t e of mutation. I f t h i s were the case, one more h i s t i d i n e r e s i d u e would be expected a t the C terminus of the 153 Apolar solvent system I 2 3 4 5 6 7 8 9 10 II 1213 P l a t e 41. A t h i n l a y e r chromatography o f DNS-amino a c i d s i n a p o l a r s o l v e n t system. 1,6,12 = V a l ; 2,8 = Asp; 3,9 = M-4-b; 4,10 = A l a ; 5,11 = u n f r a c t i o n a t e d M-4; 7,13 = M-4-a. Table XIV. Amino Acid Analysis of the Dilute Acetic Acid Cleavage of CNBr II-M Peptide M-1 M-2 M-3 M-4 M-5 M-8 M-4-a M-4-b ymol. Residues ymol. Residues umol. Residues umol. Residues ymol. Residues umol. Residues ymol. Residues Lys His Hsl* Arg Asp . 130 rhr Ser Hsr** Glu Pro Sly Ala Val Ket l i e u Leu Tyr ?he 0.017 0.26(0) 0.066 1.03(1) 0.009 0.14(0) 0.012 0.19(0) 0.066 1.03(1) 0.015 0.24(0) 0.060 0.94(1) Present .037 0.025 1.7 (2) 0.116 1.77(2) 0.075 1.73(2) 0.033 2.2 (2) 0.072 1.10(1) 0.017 1.1 (1) 0.063 0.97(1) 0.056 1.07(1) 0.047 3.1 (3) 0.200 3.06(3) 0.089 2.03 (2) 0.048 0.92(1) 0.028 1.9 (2) 0.122 1.86(2) 0.086 1.97(2) 0.016 1.1 (1) 0.063 0.97(1) Refractionated i n HVE pH 3.6 •Homoserine lactone **Homoserine U l 155 mutant peptides c o r r e s p o n d i n g t o A-5 and A-3-b from the normal a-chain. Peptides M-8 and M-5 from the mutant a-chain d i d , i n f a c t , show one more h i s t i d i n e r e s i d u e than the c o r r e s p o n d i n g amino a c i d composition o f A-5 and A-3-b r e s p e c t i v e l y (Table XIV, F i g . 40). Amino a c i d a n a l y s i s o f M-4-b was the same as M-5 except f o r the presence of one a d d i t i o n a l a s p a r t i c a c i d r e s i d u e which must be e i t h e r a t the N or the C t e r m i n a l of t h i s p e p t i d e . However, a f t e r d a n s y l a t i o n o f M-4-b, DNS-Ala was the only N t e r m i n a l amino a c i d s u g g e s t i n g t h a t the a s p a r t i c a c i d must be a t the C terminus of M-4-b ( P l a t e 41, F i g . 40). Thus, evidence from the s t u d i e s o f d i l u t e a c e t i c a c i d h y d r o l y s i s of CNBr II-A and CNBr II-M s t r o n g l y i n d i c a t e d t h a t the p o i n t mutation must have o c c u r r e d at r e s i d u e 74, a s p a r t i c a c i d b e i n g r e p l a c e d by h i s t i d i n e . To c o n f i r m t h a t h i s t i d i n e was the C t e r m i n a l amino a c i d o f M-8, a k i n e t i c study of the r e l e a s e of f r e e amino a c i d s from t h i s p e p t i d e by carboxypeptidase A-DFP was c a r r i e d out ( F i g . 42). I t may be seen t h a t h i s t i d i n e i s the amino a c i d r e l e a s e d f a s t e s t a t both 30 and 9 0 minutes o f i n c u b a t i o n at 37°C s u g g e s t i n g t h a t h i s t i d i n e must be the C t e r m i n a l amino a c i d of M-8. Thus, i n summary, s t u d i e s of d i l u t e a c e t i c a c i d cleavage products t o g e t h e r with a k i n e t i c study w i t h carboxypeptidase A on M-8 l e a d t o the c o n c l u s i o n t h a t the a s p a r t i c a c i d a t r e s i d u e 74 of the a-chain i s s u b s t i t u t e d by h i s t i d i n e ( 0 2 " ^ ^ His A 82) i n t h i s mutant. Since t h i s mutation has not been 156 F i g u r e 42. The r a t e s o f f r e e amino a c i d r e l e a s e were o b t a i n e d from the h y d r o l y s i s o f M-8 p e p t i d e by car b o x y p e p t i d a s e A. 157 d e s c r i b e d p r e v i o u s l y , i t i s proposed t h a t i t be termed Hemoglobin Mahidol. The replacement o f an a s p a r t y l by a h i s t i d y l r e s i d u e a t t h i s p o i n t , which i s l o c a t e d on the s u r f a c e o f the hemoglobin molecule at EF 3 (the 3rd r e s i d u e on the n o n - h e l i c a l segment o f EF from N terminal) would not be expected, i n case of a heterozygote, t o a l t e r the s t a b i l i t y of the hemoglobin molecule (49,77). 3. B i o c h e m i c a l evidence t o show t h a t the f a s t Hb i n S.J. A. i s a tetramer o f 8 g l o b i n c h a i n . As shown on P l a t e 32, the p a t i e n t , S.J. showed a f a s t abnormal hemoglobin upon s t a r c h g e l e l e c t r o p h o r e s i s . T h i s i s A. b e l i e v e d to be HbH(git) based on the presence o f red b l o o d c e l l i n c l u s i o n bodies and t h a l a s s e m i c changes i n r e d c e l l s (154) t o g e t h e r w i t h the o b s e r v a t i o n (made i n Bangkok) t h a t i t s m o b i l i t y i s the same as known HbH by s t a r c h g e l e l e c t r o p h o r e s i s i n t r i s - E D T A - b o r a t e b u f f e r , pH 8.6. The s t u d i e s of hemoglobin c h a i n d i s s o c i a t i o n w i t h PHMB ( P l a t e 33) and the e l u t i o n p r o f i l e o f 8-chain on the C M - c e l l u l o s e column ( F i g . 34) suggested t h a t the 8-chain o f S.J. was i d e n t i c a l w i t h 8 . Comparative s t u d i e s o f t r y p t i c p e p t i d e maps of the AEB-chain o f S.J. A showed t h a t i t was normal 8 but i t must be p r e s e n t i n excess A t o form the tetramer 8«* (HbH) . 4. Evidence f o r the i n t e r a c t i o n of a-thalassemia and Hb Mahidol A I t i s g e n e r a l l y accepted t h a t HbH ( B O i s a r e s u l t o f a-chain d e f i c i e n c y - a-thalassemia (11,12,102). S i n c e the s t a r c h g e l of S.J. shows the slow hemoglobin - Hb Mahidol and a f a s t 158 hemoglobin - b e l i e v e d t o be HbH, the p a t i e n t must have i n h e r i t e d two types of abnormal genes: the a-chain mutant - Hb Mahidol and the a-thalassemia gene which depresses the r a t e of s y n t h e s i s A A of a . Due to the absence o f a i n S.J., based on the hemo-g l o b i n c h a i n d i s s o c i a t i o n study with PHMB ( P l a t e 33) and CM-c e l l u l o s e chromatography ( F i g . 34) there i s a t o t a l absence of HbA as shown i n s t a r c h g e l e l e c t r o p h o r e s i s ( P l a t e 32). T h i s suggests t h a t the a-thalassemia gene may be an a l l e l i c or c l o s e l y l i n k e d t o t h a t of the a-chain v a r i a n t - Hb Mahidol and t h a t s t r o n g i n t e r a c t i o n has o c c u r r e d so as t o depress s e v e r e l y A A the a c t i v i t y o f the a l o c u s . The absence of a would l e a d A A to some f r e e 8 which would then polymerise t o 6* (HbH). The g e n e t i c a l f i n d i n g s ( F i g . 31 and T a b l e X) a l s o confirmed t h a t the double h e t e r o z y g o t e form of a-thalassemia and the Hb Mahidol i n S.J. p a t i e n t as evidenced by the s e g r e g a t i o n of both abnormal genes i n h i s sons, namely a-thalassemia t r a i t i n I I I - 2 and Hb Mahidol t r a i t i n I I I - l , w h i l e h i s w i f e I I - l was not thought t o possess an abnormal gene. Furthermore, the a-thalassemia t r a i t was a l s o found to be p r e s e n t i n h i s mother 1-2. F u r t h e r evidence f o r i n t e r a c t i o n o f these two genes was p r o v i d e d by c l i n i c a l and h e m a t o l o g i c a l s t u d i e s . In the case of the h e t e r o z y g o t e , e i t h e r a-thalassemia i n 1-2, I I I - 2 , or Hb Mahidol i n I I I - l , both appeared h e a l t h y . But the double heterozygote S.J. had e v i d e n t l y s u f f e r e d from c h r o n i c anemia and jaundice s i n c e c h i l d h o o d . The c l i n i c a l and h e m a t o l o g i c a l f e a t u r e s , as d e s c r i b e d p r e v i o u s l y , a l s o r e v e a l e d a syndrome 159 o f c h r o n i c hemolysis w i t h severe t h a l a s s e m i c a b n o r m a l i t i e s i n r e d b l o o d c e l l s . A l l these consequences must have r e s u l t e d from the i n t e r a c t i o n of both abnormal genes. I t i s w e l l known t h a t the i n t e r a c t i o n o f a g-chain v a r i a n t w i t h a g-thalassemia gene, presumably a t an a l l e l i c or c l o s e l y l i n k e d l o c u s , can m a n i f e s t i t s e l f c l i n i c a l l y as a severe d e p r e s s i o n of g s y n t h e s i s , e.g. g-thalassemia-HbE, g-thalassemia-Hbs,S, g-thalassemia-HbC (12). In c o n t r a s t , only a few cases o f a-chain mutant a s s o c i a t i o n w i t h a-thalassemia, namely a-thalassemia-HbQ (155,156,157,158) and a-thalassemia-Hbl (159) have been d e s c r i b e d . U n f o r t u n a t e l y , b i o c h e m i c a l study o f HbQ, has not y e t r e v e a l e d the nature of the mutation. Our case, S.J., shown above t o be a-thalassemia-Hb Mahidol, i s s i m i l a r t o a-thalassemia-HbQ i n terms of having HbH, a slow HbA2 which probably r e p r e s e n t s a 2 M u t a n t 6 A 2 and a t o t a l absence of HbA. 5. B i o c h e m i c a l s t u d i e s t o show t h a t S.T. and S.R. are i d e n t i c a l t o Hb Mahidol The heterozygote of S.T. and S.R. samples ( P l a t e 32,33) were a l s o i n v e s t i g a t e d . G l o b i n f r a c t i o n a t i o n by C M - c e l l u l o s e chromatography (74) o f both sample showed the same r e s o l u t i o n F i g . 43. I s o l a t i o n of the a-chain of both were c a r r i e d out i n the same manner as f o r the S.J. sample. A comparison of the t r y p t i c p eptide maps o f normal and v a r i a n t a-chains showed t h a t the s i t e o f mutation o c c u r r e d at aTpIX. Furthermore, the trypsin-CNBr c l e a v e d p e p t i d e map i n d i c a t e d t h a t the amino a c i d F i g u r e 43. C M - c e l l u l o s e chromatography o f S.R. g l o b i n (600 mg) i n 8M u r e a , 0.05M 8-mercaptoethanol and sodium phosphate b u f f e r . 161 a l t e r a t i o n was l o c a t e d i n the C N B r l l peptide ( r e s i d u e 62-76 o f a - c h a i n ) ( F i g . 37). The p e p t i d e s , CNBrll-M o f S.T. and S.R. l o c a t e d by l i g h t n i n h y d r i n s t a i n i n g of the p e p t i d e maps were e l u t e d w i t h 6N HCl and h y d r o l y z e d . The amino a c i d analyses of both are shown i n T a b l e XV and XVI. The replacement of an a s p a r t i c a c i d or asparagine by h i s t i d i n e was suggested i n both cases. High v o l t a g e e l e c t r o p h o r e s i s o f the d i l u t e a c e t i c h y d r o l y s a t e s o f i s o l a t e d CNBrll-M from S.T. and S.R. were i d e n t i c a l t o t h a t of the S.J. sample f o r the 8 fragments, r e l a t i v e t o the amino a c i d markers ( P l a t e 44). T h e r e f o r e , the amino a c i d a l t e r a t i o n must be the same as f o r S.J. i n which the a s p a r t y l r e s i d u e o f 74 i s r e p l a c e d by h i s t i d i n e . The amino a c i d compositions of the key p e p t i d e s , M - l , M-2, M-3 and M-8 of both cases were s t u d i e d (Table XVII and X V I I I ) . I t may be seen t h a t i n a l l cases M-l, M-2, M-3 and M-8 gave f r e e a s p a r t i c a c i d , Ala-Leu-Thr f o r r e s i d u e 65-67, f r e e homoserine which e q u i l i b r a t e t o homoserine l a c t o n e and A l a - V a l - A l a - H i s - V a l - H i s f o r r e s i d u e 69-74 r e s p e c t i v e l y ( F i g . 45). In c o n c l u s i o n , the S.T. and S.R. samples were found t o be a heterozygote f o r Hb Mahidol ( a J " H l s 3 A ) . C. Summary A slow ( l e s s a n i o n i c ) hemoglobin mutant has been d e t e c t e d by s t a r c h g e l e l e c t r o p h o r e s i s o f hemoglobin from three u n r e l a t e d p a t i e n t s i n Bangkok. D i s s o c i a t i o n o f the abnormal hemoglobin wit h PHMB showed t h a t the a-chain was the s i t e o f mutation. 162 C N B r I I - M S .R, | ** A m i n o acid m o r k e r s C N B r U M S,T, , S T A n o d e C N B r l - M S . J , Amino a c i d m a r k e r s C N B r H A — N 10 » m 10 N oo ) I I I I I I Z Z Z S Z Z Z 2 P l a t e 44. Guide s t r i p s o f d i l u t e a c e t i c a c i d h y d r o l y s a t e of CNBrll-M of S.T. and S.R. sample analyzed by paper e l e c t r o p h o r e s i s pH 1.9 with cadmium n i n h y d r i n s t a i n . The m o b i l i t y o f fragments o f CNBrll-M-SR and CNBrll-M-ST were i d e n t i c a l t o CNBrll-M-S.J. S.J. Sample (Hb Mahidol) CNBrll-M-62 64 68 74 75 76 V a l - A l a - Asp - Ala-Leu-Thr - Asn - A l a - V a l - A l a - H i s - V a l - H i s - Asp - H s l -«-M- 4- a A-1 M-2 >(<-A-l->|« M-8 •|«-M-l-H*-M-3» f< • M-5 >| |< M-4-b * f S.T. and S.R. Sample CNBrll-M' 62 64 68 " 74 75 76 V a l - A l a - Asp - Ala-Leu-Thr - Asn - A l a - V a l - A l a - H i s - V a l - H i s - Asp - H s l | < - M - 1 - » | « — M - 2 ? - ) < - M-l>)<= M - 8 > | - < - M - l » f ^ r M - 3 4 F i g u r e 45. A diagram of amino a c i d analyses o f d i l u t e a c e t i c c l e a v e d fragments o f CNBrll-M of S.J. (Hb Mahidol) and S.T. & S.R. sample. 164 T a b l e XV. Amino a c i d a n a l y s i s o f C N B r l l - M o f S.T. sample w h i c h was e l u t e d w i t h 6N HCl from 5 mg o f t r y p s i n - C N B r c l e a v a g e <5f p e p t i d e map a f t e r l i g h t n i n h y d r i n s t a i n i n g . CNBrII-M C N B r l l - A umoles r e s i d u e s t h e o r e t i c a l r e s i d u e s Lys H i s 0.109 1.70 (2) 1 H s l * P r e s e n t A r g Asp 0.208 3.23 (3) 4 Thr 0.069 1.08 (1) 1 Se r H s r * * 0 .020 0.31 (1) 1 G l u P r o G l y A l a 0.281 4.30 (4) 4 V a l 0.171 2.70 (3) 3 Met Leu 0 .079 1.20 (1) 1 T y r Phe * H s l = Homoserine l a c t o n e **Hsr = Homoserine 165 T a b l e X V I . Amino a c i d a n a l y s i s o f CNBrII-M o f SR sample was o b t a i n e d from 5 mg o f t r y p s i n - C N B r t r e a t e d p e p t i d e map by d i r e c t 6N HCl e l u t i o n a f t e r l i g h t n i n h y d r i n s t a i n i n g . CNBrII-M C N B r l l - A umoles r e s i d u e s t h e o r e t i c a l r e s i d u e s Lys Hi s 0.10 0 1.60 (2) 1 H s l * P r e s e n t Arg Asp 0.19 8 3.10 (3) 4 Thr 0.070 1.10 (1) 1 Ser H s r * * 0.025 0.40 (1) 1 G l u P r o Gly A l a 0.266 4.20 (4) 4 V a l 0.174 2.76 (3) 3 Met Leu 0.074 1.10 (1) 1 T y r Phe * H s l = Homoserine l a c t o n e **Hsr = Homoserine 166 Table XVII. Amino a c i d analyses o f 0.25M a c e t i c a c i d h y d r o l y s i s fragments o f CNBrll-M of S.T. sample. M-l M-2 M-3 M-8 ymoles r e s i d u e s ymoles r e s i d u e s ymoles r e s i d u e s ymoles residue Lys His 0.033 1.81 C H s l Present f r e e H s l Arg Asp 0.060 f r e e Asp Thr 0.032 0.99 (1) Ser Hsr 0.040 f r e e Hsr Glu Pro Gly A l a 0.035 1.09 (1) 0.039 2.17 C V a l 0.036 2.00 C Met Leu 0.029 0 .91 (1) Tyr Phe 167 Table XVIII. Amino a c i d analyses o f d i l u t e a c e t i c cleavage fragments o f CNBrII-M cleavage o f S.R. sample. M-1 M-2 M-3 M-8 umoles r e s i d u e s umoles r e s i d u e s umoles r e s i d u e s umoles residue Lys His 0.047 1.96 C Hsl P r e s e n t f r e e H s l Arg Asp 0.070 f r e e Asp Thr 0.034 1.03 (1) Ser Hsr 0 .050 f r e e Hsr Glu Pro Gly A l a 0.036 1.09 (1) 0.055 2.20 C V a l 0.044 1.83 0 Met Leu 0.031 0 .94 (1) Tyr Phe 168 The mutant a-chain was i s o l a t e d by C M - c e l l u l o s e chromatography i n 8M urea and 0.05M g-mercaptoethanol. P e p t i d e maps of t r y p s i n and cyanogen bromide c l e a v e d a-chains i n d i c a t e d t h a t the amino a c i d a l t e r a t i o n o f the mutant was i n the p e p t i d e c o r r e s p o n d i n g to r e s i d u e s 6 2-76 of a-chain. F u r t h e r cleavage of t h i s p e p t i d e w i t h 0.25M a c e t i c a c i d a t 110° C showed t h a t r e s i d u e 74 was changed from a s p a r t y l t o a h i s t i d y l r e s i d u e , a mutation not p r e v i o u s l y d e s c r i b e d . I t i s proposed t h a t t h i s new hemoglobin aji»HiSgA ^ c a3_j_ ecl hemoglobin Mahidol a f t e r Mahidol U n i v e r s i t y i n Bangkok. One o f the t h r e e p a t i e n t s , S.J. showed the combination o f a-thalassemia and Hb Mahidol. T h i s evidence was a l s o confirmed by the g e n e t i c a l s t u d i e s of s e g r e g a t i o n of both genes i n h i s sons. The i n t e r a c t i o n of a-thalassemia and Hb Mahidol r e s u l t e d i n the appearance of the c l i n i c a l syndromes of c h r o n i c h e m o l y t i c anemia i n the S.J. p a t i e n t s i n c e c h i l d h o o d . The h e m a t o l o g i c a l f i n d i n g s a l s o i n d i c a t e d c h r o n i c hemolysis w i t h severe t h a l a s s e m i c r e d c e l l a b n o r m a l i t i e s . 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